TWI523867B - Anti-c-met antibody and methods of use thereof - Google Patents

Description

Anti-C-MET antibody and method of use thereof

The present invention relates to antibodies that bind to c-Met, as well as related compositions, and methods of use thereof.

In the United States, lung cancer is the leading cause of death in men and women from cancer. In 2009, an estimated 219,000 new cases of lung cancer were diagnosed in the United States, and approximately 160,000 deaths were caused by the disease. Two types of lung cancer, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), which cause nearly 80% of lung cancer, are known. The five-year survival rate for patients with NSCLC is approximately 16%. Although chemotherapy, surgical resection, and radiation therapy have been used to treat different stages of NSCLC, the prognosis is still unsatisfactory and may recur after initial treatment, which may be as high as 10%.

c-Met is a receptor for hepatocyte growth factor and belongs to the subfamily of receptor tyrosine kinases (RTKs). In general physiological conditions, the HGF/c-Met pathway is involved in many different biological functions, including cell proliferation, survival, activity, and healing (Birchmeier et al., 2003). However, in clinical cases of hematological cancers and most solid tumors, abnormal c-Met activation, including gene amplification, mutation and overexpression, can be found. c-Met activation has been reported to cause cancer cell proliferation, migration and invasion, and to promote tumor angiogenesis due to HGF directly stimulating endothelial cell proliferation and migration.

In addition, in patients with brain cancer, colorectal cancer, stomach cancer, lung cancer, head and neck cancer, and gastric cancer, excessive expression of c-Met is often observed. Poor clinical outcomes are clearly associated with elevated c-Met, so in these cancer types, it is speculated that overexpression of c-Met is a negative prognostic indicator of cancer progression.

Antibodies that bind to c-Met, as well as related compositions, and methods of use thereof, are disclosed herein. Methods of use include, but are not limited to, cancer treatment and diagnosis. In some embodiments, an antibody of the invention binds to a mammalian cell surface antigen (eg, a cancer cell surface antigen). The antibody can also be pinched to bind to cells. The cells that can be targeted by the antibody include cancer cells, such as cancer cells equal to lungs, kidneys, liver, stomach, breasts, and brain.

In particular, the antibodies of the invention may specifically bind to the epitope of c-Met. The epitope can be defined by its location between the SEMA region and the PSI region, or within the IgG-like region of c-Met. More specifically, the epitope comprises an amino acid of SEQ ID NO: 51 (residue position 25-567 of c-Met) or SEQ ID NO: 52 (residue position 567-932 of c-Met) sequence. The c-Met residue position number used above corresponds to the sequence contained in GenBank Accession No. NP_000236.2 (SEQ ID NO: 53) or UniProt Accession No. P08581.

In some embodiments, the antibodies of the invention can be:

(a) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region ( _VH ) comprising a complementarity determining region (CDR) 1, CDR2, and a CDR3 sequence, respectively An amino acid sequence comprising SEQ ID NOS: 10, 12 and 22;

(b) an antibody or antigen-binding fragment thereof, comprising a light chain variable region (V _L), which is complementary to the light chain variable region comprising CDR1, CDR2, and CDR3 sequences, respectively, comprising SEQ ID NOS: 31,33 and 43 Amino acid sequence;

(c) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region ( _VH ) comprising CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOS: 10, 12, and 22, respectively the amino acid sequence, and a light chain variable region (V _L), which is complementary to the light chain variable region comprising CDR1, CDR2, and CDR3 sequences, respectively, comprising SEQ ID NOS: 31, 33 and 43 of the amino acid sequence;

(d) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region ( _VH ) comprising CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOS: 14, 16 and 25, respectively Amino acid sequence;

(e) an antibody or antigen-binding fragment thereof, comprising a light chain variable region (V _L), which is complementary to the light chain variable region comprising CDR1, CDR2, and CDR3 sequences, respectively, comprising SEQ ID NOS: 35,37 and 46 Amino acid sequence;

(f) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region ( _VH ) comprising CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOS: 14, 16 and 25, respectively the amino acid sequence, and a light chain variable region (V _L), which is complementary to the light chain variable region comprising CDR1, CDR2, and CDR3 sequences, respectively, comprising SEQ ID NOS: 35, 37 and 46 of the amino acid sequence;

(g) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region ( _VH ) comprising CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOS: 18, 20, and 28, respectively Amino acid sequence;

(h) an antibody or antigen-binding fragment thereof, comprising a light chain variable region (V _L), which is complementary to the light chain variable region comprising CDR1, CDR2, and CDR3 sequences, respectively, comprising SEQ ID NOS: 39,41 and 49 Amino acid sequence; or

(i) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region ( _VH ) comprising CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOS: 18, 20, and 28, respectively the amino acid sequence, and a light chain variable region (V _L), which is complementary to the light chain variable region comprising CDR1, CDR2, and CDR3 sequences, respectively, comprising SEQ ID NOS: 39, 41 and 49 of the amino acid sequence.

definition

In the following description, there are many terms that are conventional and widely used in the field of cell culture. To the extent that the specification and claims are made clear and consistent, the scope and definition of such terms are provided below.

As used herein, "c-Met" refers to a group of receptor tyrosine kinases that bind to hepatocyte growth factor (HGF) and is also known as hepatocyte growth factor receptor (hepatocyte growth factor receptor). HGFR) or "met proto-oncogene". The word "c-Met" refers to any isoform of the naturally occurring c-Met protein. The amino acid sequence of c-Met is known and can be found in GenBank Accession Nos. NP_000236.2 and NP_001120972.1.

The terms "polypeptide", "peptide", or "protein" are used interchangeably herein to refer to a linear sequence of amino acid residues which are mutually dependent on each other by the alpha amine group and the adjacent residue of the carboxyl group. The peptide bond between the peptides. In addition, the amino acid includes an amino acid analog in addition to the amino acid encoded by 20 "standard" genes.

An "antibody" comprises a composition comprising an antigen binding protein, or as a preparation comprising a plurality of antigen binding proteins having one or more multi-peptides, which may be genes of immunoglobulin genes A fragment of the encoded, or immunoglobulin gene, or a CDR thereof obtained or derived from a library of phage expression, and which binds to the desired antigen. Light chains are classified as κ (kappa) or λ (lambda). Heavy chains can be divided into gamma (gamma), μ (mu), alpha (alpha), δ (delta), or ε (epsilon), which can be used to define the types of immunoglobulins, IgG, IgM, IgA, IgD and IgE.

One example of an antibody is a structural unit having a tetramer consisting of two pairs of multi-peptide chains, each pair having a "light" and a "heavy" chain. The N-terminal portion of each chain is a variable region that regulates antigen binding. The terms "variable light chain (V _L )" and "variable heavy chain (V _H )" refer to both light and heavy chains, respectively.

An "antibody" also encompasses a single chain antibody comprising a heavy chain and a light chain that are joined to each other to form a single multi-peptide.

As described above, the "antibody" comprises an intact immunoglobulin or may be an antigen-binding fragment of an antibody. Thus, the term "antibody" as used herein also includes an antigen binding portion of an antibody which can be made by modifying the whole antibody or by using recombinant DNA methods to synthesize de novo . Examples include, but are not limited to, Fab', Fab' ₂ , or scFv.

A single chain Fv ( "scFv") is a multi-peptide V _H :: V _L heterodimer covalently coupled to, the performance of which may be from a nucleic acid, the nucleic acid comprising a V _H - and V _L - encoding sequences, the coding sequence It can be directly linked or linked by a peptide-encoding linker. There are many structures available for light and heavy multi-peptide chains, from the antibody V region, to the scFv molecule, which will fold into a three-dimensional structure that is essentially similar in structure to the antigen binding site. In addition to being a diabody, the scFvs can also be used as a tribody or a tetrabody.

It is to be understood that when different antibody fragments are defined by cleavage digestion of intact antibodies, it will be apparent to those of ordinary skill in the art that such fragments can be resynthesized by chemical or recombinant DNA methods.

The term "antibody" includes a plurality of antibodies and monoclonal antibodies, and further comprises any type of antibody (eg, IgM, IgG, and subtypes thereof). "Antibody" also includes a hybrid antibody, a heteroantibody, a chimeric antibody, a humanized antibody, and functional fragments thereof, which retain antigen binding ability. The antibody may bind to other molecular bodies and/or may be bound to a support (eg, a solid support) such as a polystyrene disk or bead, a test strip, and the like.

An immunoglobulin light chain variable region or heavy chain variable region is composed of a framework region (FR), which is distinguished by three hypervariable regions, also known as hypervariable regions. As a "complementary decision area" or "CDRs". The length of the architectural region and CDRs can be defined by a known database. See, for example, V Base at www.vbase2.org. Sequences of the framework regions of different light or heavy chains are relatively preserved in the species. The framework region of the antibody, which is the framework region that constitutes the combination of the light and heavy chains, can be used to position and align the CDRs. The CDRs are primarily responsible for binding to the epitope of the antigen. Unless otherwise indicated, all CDRs and architectural regions provided by the present invention are as defined by V Base.

"Anti-c-Met antibody" refers to an antibody that specifically binds to c-Met and preferably has a high affinity to c-Met. A c-Met-specific antibody does not bind to other c-Met-independent antigens, but only to c-Met binding.

When the term "high affinity" is used in the context of an antibody, it is meant that the antibody specifically binds (identifies) to its target having less than or equal to 10 ^-6 M, less than 10 ^-7 M, less than 10 ^-8 M. Affinity value (K _D ). A lower K _D value means higher binding affinity (eg, stronger binding capacity), therefore, a higher binding affinity of 10 ^-7 K _D values than a 10 ^-6 K _D value .

"Antigen binding site" or "binding moiety" refers to a portion of an antibody molecule (eg, an immunoglobulin molecule or a fragment of an scFv) that is involved in antigen binding by an immune response. The antigen binding site is formed by an amino acid residue of the N-terminal variable region (V) of the heavy (H) chain and/or the light (L) chain. The three highly divergent branches in the V zone of the heavy and light chains are "hypervariable zones" which are interposed between the more conservative lateral branches, the conservative measurement Branches are also known as "architectural areas" or "FRs". Therefore, the term "FR" refers to an amino acid sequence naturally present between and adjacent to the hypervariable region of an immunoglobulin. In a tetrameric antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in three dimensions and arranged opposite each other to form an antigen-binding "surface". The surface regulates the recognition and binding of the target antigen. The three hypervariable regions of each heavy chain and/or light chain are "complementarity determining regions" or "CDRs".

An "antigenic determinant" is a site on an antigen (eg, a site on the c-Met Sema or PSI domain) that is available for antibody binding. The epitope can be formed by a continuous amino acid or a discontinuous amino acid and can be juxtaposed by protein folding (eg, tertiary folding).

"S21 antibody" or "antibody obtained from self-selected strain 21" refers to an antibody which is expressed by a strain S21 or a strain 21 or is synthesized by other means, but is still associated with the selected strain S21. The antibodies expressed have the same CDRs and, optionally, the same framework regions. Similarly, antibodies S1 (selection strain 1) and S20 (selection strain 20), and analogs thereof, mean that the resistance system is expressed by a corresponding selection strain, and/or otherwise synthesizes antibodies, but It still has the same CDRs as the corresponding antibody and, optionally, has the same framework region. See CDRs for these antibodies as shown in Table 1 below.

As used herein, the terms "subject," "individual," and "patient" are used interchangeably to refer to a mammal that is evaluated for treatment and/or treatment. In a specific embodiment, the mammal is a human. The terms "subject", "individual" and "patient" include individuals with cancer (eg, adenocarcinoma of lung cancer, ovary or prostate, breast cancer, etc.). The subject can be human, but also includes other mammals, especially mammals that can be used in laboratory models for studying human diseases, such as mice, rats, and the like.

The terms "treatment", "treating" and the like, as used herein, refer to administration of a medicament, or a surgical procedure (eg, radiation, surgery, etc.) for the purpose of achieving efficacy. The efficacy may be prophylactic, such as completely or partially preventing the disease or its symptoms, and/or may be therapeutically, such as partially or completely curing the disease, and/or the symptoms of the disease. As used herein, "treatment" encompasses the treatment of any proliferative growth in a mammal, particularly a human, and includes: (a) preventing the individual from being afflicted with the symptoms of the disease or condition, the individual may be susceptible to the disease However, it has not been diagnosed as having (eg, including a disease associated with a primary disease or a disease caused by a primary disease); (b) inhibiting the disease, such as stopping the development of the disease; (c) mitigating Diseases, such as: make the disease condition better. In the treatment of tumors (eg, cancer), therapeutic agents can directly reduce the metastasis of tumor cells.

The term "cell culture" or "culture" refers to maintaining cell growth in an artificial or in vitro environment. Although the term "cell culture" is a generic term, it is also understood that the term encompasses cultures, not just individual cells, but also tissues or organs.

The term "tumor" as used herein refers to the growth and proliferation of all tumor cells, whether malignant or benign, and includes all precancerous, cancerous cells and tissues.

As used herein, the terms "cancer," "neoplasm," and "tumor" are used to refer to cells that have spontaneous, uncontrolled growth; such cells are detached from normal growth. The type is characterized in that its overall cell proliferation is clearly out of control. In general, the cells of the invention to be detected, analyzed, graded, or treated include pre-cancerous (eg, benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. Examples of cancer include, but are not limited to, lung cancer, kidney cancer (eg, renal cancer), gastric cancer, breast cancer, brain cancer, lung cancer, prostate cancer, hepatocellular carcinoma, pancreatic cancer, cervical cancer, ovarian cancer, Liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, tumor, melanoma, head and neck cancer, and colorectal cancer.

According to the nature of the cancer, to obtain a sample of the appropriate patient. The term "cancer tissue sample" as used herein refers to any cell obtained from a cancerous tumor. In the case of solid tumors, tissue samples are typically taken from surgically removed tumors and tested by conventional techniques. Alternatively, a body fluid sample, such as a lymphatic, blood or serum sample, or a oozing liquid sample, such as a cancer organ exudate (eg, breast exudate), can be collected and used as a sample for analysis. In the case of blood cancer, lymphocytes or leukemia cells can be obtained and treated in an appropriate manner. Similarly, in the case of any metastatic cancer, the cells can be obtained from a bodily fluid such as lymph, blood, serum, or distally infected organs or exudates thereof.

The "pathology" of cancer includes all phenomena that endanger the health of the patient. This includes, but is not limited to, abnormal or uncontrolled cell growth, metastasis, interference with the normal function of adjacent cells, release of cytokines or other released products with abnormal levels, inhibition or deterioration of inflammation or immune response, tumor formation, Initial cancer, malignant tumor, infiltration of peripheral or distal tissues or organs, such as lymph nodes.

As used herein, the term "diagnosis" refers to the identification of a molecular or pathological condition, disease or condition, such as a molecular subtype that identifies breast cancer, prostate cancer, or other types of cancer.

The term "prognosis" as used herein refers to the possibility of predicting the development of cancer death or cancer, including recurrence, metastatic spread, and drug resistance of tumor diseases such as lung cancer, colorectal cancer, skin cancer or esophageal cancer. The term "forecast" as used herein refers to a prediction or estimate based on observations, experience, or scientific reasons. In one example, the physician can predict the likelihood that the patient will survive when the patient has surgically removed the primary tumor and/or after a period of chemotherapy without a cancer recurrence.

The term "related" or "related" and other similar terms as used herein refers to the statistical correlation of two events, including the number, data set, and the like. For example, when the event involves a number, positive correlation (also referred to herein as "direct correlation") means that when one increases, the other increases. Negative correlation (also referred to herein as "reverse correlation") means that when one increases, the other decreases.

The term "isolated" is intended to indicate that a compound is isolated from all or part of the components naturally associated with the compound. "Isolation" also refers to a compound (eg, a protein) that is separated from all or part of its concomitant components during the manufacturing process (eg, chemical synthesis, recombinant expression, culture medium, and the like).

A "biological sample" consists of different sample types taken from an individual. The definition includes liquid samples of blood and other biological organs, solid tissue samples (eg, living samples, or tissue culture, or cells obtained from them, and their progeny). ). The definition also includes samples that have been processed in any manner since they were obtained, such as treatment, washing, or enrichment of specific cell populations (eg, cancer cells). The definition also includes samples that are enriched for a particular molecular type, such as nucleic acids, multi-peptides, and the like. The term "biological sample" includes clinical samples, as well as tissues from surgical resection, tissues obtained from biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, Serum and its analogues. "Biological sample" includes samples obtained from cancer cells of a patient, such as: a polynucleotide containing a polynucleotide and/or a multi-peptide, which is derived from a patient's cancer cells (eg, cell lysates or other polynucleotides and/or Or a cell extract of a multi-peptide, obtained; and the same cancer cells contained in the patient. A biological sample contains cancer cells from a patient and may also include non-cancer cells.

Description of specific embodiments

The anti-system disclosed herein is a specific binding partner to c-Met, and compositions related thereto and methods of use thereof. Methods of use include cancer therapy and diagnosis.

The antibody comprises at least one, two or all three CDRs, the CDRs is derived from an antibody V _H clones are 21, 20 or of the. The antibody also includes those comprising one, at least two or all three CDRs, the CDRs is derived from an antibody V _L clones are 21, 20 or of the. Each CDR V _H or V _L can be selected independently of. Alternatively, the antibody system competes with the antibody derived from the selected strain 1, 20 or 21 for binding to c-Met (eg, binding to the same epitope).

Antibody of the present invention can also include all V _H CDRs and / or V _L CDRs is derived from an antibody clones are 21, 20 or of the. The antibody may comprise a full length _VH chain derived from an antibody of the selected strain 1, 20 or 21. The antibody may also comprise a full-length clones are optional source V _L chain of antibody 1.20 or 21.

The antibody may be a single chain Fv (scFv), Fab, (Fab') ₂ , (ScFv) _2, and the like. The antibody may be an IgG (eg, IgG ₂ ) or other isotype, or may be a pair of specific antibodies.

The antibody can be linked to, for example, an anticancer drug, a tag, a small molecule (e.g., PEG) that improves serum half-life, a pinocytosis, and the like. The antibody may also be a pharmaceutically acceptable excipient (e.g., present in a unit dosage formulation). The invention also provides compositions comprising one or more different antibodies selected from the antibodies described herein, and/or antibodies comprising one or more CDRs derived from the antibody, and/or one or more An antibody comprising a mutated or derivatized antibody. The composition may include one or more antibodies, such as a selection strain 1, 20, or 21.

The methods of the invention comprise administering one or more of the antibodies described herein, in an amount effective to treat a subject having cancer, wherein the antigen exhibited by the cancer is conjugated by the antibody. The antibodies provided by the present invention can also be used to diagnose/prognose the development of cancer.

The nucleic acid provided herein encodes one or more of the antibodies described herein. Host cells comprising such nucleic acids are also provided herein, and such cells can produce such antibodies (e.g., by secretion). The invention also provides a kit for preparing a composition comprising the antibody, or for performing the methods described.

antibody

Preferred antibodies have a high affinity for c-Met, a cell membrane receptor that can be exposed to the cell surface of cancer cells. Cancer cells include, for example, those derived from lung cancer cells (eg, H1993 or H441) and other cancer cells. The antibody includes the antibody which is internalized into the cell after binding to the antigen, for example, an antigen located on the surface of a living mammalian cell, such as a pinocytosis effect, such as a pinocytosis of a receptor vector.

The antibody includes an antibody that competes with the antibody derived from the selected strain 1, 20, or 21 and competes for binding to the c-Met epitope. The ability of a particular antibody to recognize the same epitope as other antibodies can be determined by the ability of an antibody to compete for inhibition of binding of the second antibody to the antigen (e.g., by competition binding assays). The antibodies described herein also comprise an antibody that binds to the same epitope as the antibody from the selected strain 1, 20, or 21.

Any competitive binding assay that can be used to measure the binding of two antibodies to the same antigen can be used. For example, a sandwich ELISA test can achieve this. Methods for analyzing cross-reactivity are well known to those of ordinary skill in the art (see, for example, Dowbenko et al. (1988) J. Virol . 62: 4703-4711).

When the first antibody is used in any assay for competitive binding assays, if the ability of the second antibody to bind to the antigen is reduced by at least 30%, typically at least about 40%, 50%, 60% or 75%, and usually at least At about 90%, an antibody is considered to competitively inhibit binding of the second antibody.

An antibody binding can also be analyzed by providing one or more isolated target antigens (eg, full length c-Met or fragments thereof) for attachment to a solid support (eg, using surface plasma resonance). The ability to the target, or the ability to compete with the antibodies described herein for binding to the target, is determined.

An epitope that can be bound by an anti-c-Met antibody (eg, strains 1 and 20) is present at a binding site of a c-Met ligand (eg, hepatocyte growth factor). The binding site of hepatocyte growth factor is located at the 25-567 residue position of the linked amino acid sequence of c-Met. The epitope can also be defined by its location in the SEMA and PSI intervals.

Furthermore, the epitope to which the anti-c-Met antibody (e.g., the selection strain 21) binds may be present from about 567 residues to about 932 residues of the linked amino acid sequence of c-Met. The epitope can also be defined by its position in the IgG region such as c-Met.

The c-Met residue position number used above is based on the sequence described in GenBank Accession No. NP_000236.2 or UniProt Accession No. P08581.

An antigen having the same epitope as c-Met is also the subject of binding of the antibodies described herein. When bound to c-Met, the antibody can be internalized by cells that express the c-Met protein.

The anti-c-Met antibody binds affinity to the epitope and is cell surface exposed and solvent-accessible on many cancer cells, especially on the cell membrane of cells. When the cell is alive, the epitope can be contacted by the antibody. For example, the epitope may be present on cancer cells derived from the lungs, kidneys, liver, stomach, breasts, and brain. The anti-c-Met antibody can bind to cancer cells affinityly, and can be any cancer containing c-Met cancer cells.

As described above, the antibody comprises an antibody that competes with one or more antibodies derived from a selection strain 1, 20, or 21 or the like. In addition, the antibody may have a binding affinity of, based equivalent to or greater than a c-Met antibodies having from about 1 x K _D value of 10 ^-6 M. The K _D value of the antibody of the present invention for c-Met may range from 1 × 10 ^-6 M to about 1 × 10 ^-7 M, and is between about 1 × 10 ^-7 M and about 1 × 10 ^-8 . It is from about 1 x 10 ^-8 M to about 1 x 10 ^-9 M. For example: the range of K _D values of antibodies of the invention can be between about 5 × 10 ^-9 M to about 2 × 10 ^-8 M.

Examples of such antibodies comprise the same binding specificity, and comprise at least two CDRs, each independently and with each of the antibodies shown in Table 1 below (eg, _VH CDR1 of the selected strain 21) The amino acid sequence of the _VH CDRs has at least about 80%, at least about 87%, at least about 93%, at least about 94%, or up to 100% amino acid sequence identity. The antibody may also include all three CDRs from any of the _VH CDRs of each of the antibodies shown in Table 1; wherein each _VH CDR of the antibody is selected from the single antibodies shown in Table 1, and each The _VH CDRs independently, with at least about 80%, at least about 87%, at least about 93%, at least about 94%, or up to 100% of the amino group of the _VH CDRs of the antibodies set forth in Table 1, Acid sequence identity. For example, the heavy chain of the antibody may comprise two _VH CDRs or all three _VH CDRs of the selected strain 21. Alternatively, the heavy chain may comprise two _VH CDRs or all three _VH CDRs of the selected strain 21.

The light chains are also similar, the antibodies will have the same binding specificity, and may comprise at least two CDRs, each independently and with each of the antibodies shown in Table 1 below (eg, the selection of strain 21) The amino acid sequence of the _VL CDR of _VL CDR1) has at least about 80%, at least about 87%, at least about 93%, at least about 94%, or up to 100% amino acid sequence identity. The antibodies may also include any from the table shown in all three V _L CDRs of an antibody, and independently of each V _L CDR with the amino acid sequence V _L CDR of an antibody shown in Table 1, having at least about 80%, at least about 87%, at least about 93%, at least about 94%, or up to 100% amino acid sequence identity. For example: the light chain of antibody 21 may comprise two V _L CDRs, or all three clones are V _L CDRs. Or, 21 may comprise a light chain V _L CDRs of two or all three clones are V _L CDRs.

Alternatively, the antibody of the present invention may comprise the same (e.g., 100% identical), similar or different architectural sequence (FR) as the framework sequence corresponding to the heavy or light chain provided in Table 1. When the sequence of the architecture is similar, the framework sequence corresponding to any of the antibodies shown in Table 1 below can have at least about 85%, at least about 86%, at least about 90%, at least about 93%, at least about 96%, at least about 98%, or up to 100% identity.

Accordingly, the antibodies of the present invention may comprise V _H and / or V _L sequences of the full-length full-length, full length thereof as shown in Table 1 of the V _H or V _L sequence having at least 80% identity, at least 85%, at least 90 %, at least 95%, or up to 100% amino acid sequence identity. For example: The antibody may comprise full-length clones are of V _H 21 and / or the full length V _L. Or, the antibody may comprise full-length clones are of V _H 20 and / or the full length V _L.

Method of producing antibodies

Using the information provided herein, the anti-c-Met anti-system of the present invention is prepared in accordance with standard techniques well known to those of ordinary skill in the art. For example, the multi-peptide sequence provided herein (see, eg, Table 1) can be used to determine the appropriate nucleic acid sequence to encode the antibody, and then the nucleic acid sequence exhibits one or more specificity for c-Met. antibody. The nucleic acid sequences can be adapted to present a particular codon to "cater" to the codons required for the different expression systems, according to standard methods well known in the art to those skilled in the art.

Using the sequence information provided, the nucleic acid can be synthesized according to a number of standard methods well known in the art to those skilled in the art. Oligonucleotide synthesis is preferably synthesized by commercially available solid phase oligonucleotide synthesis machinery, or artificially synthesized using, for example, a solid phase phosphite triester method.

Once the nucleic acid encoding the antibody is synthesized, it can be amplified and/or cloned according to standard methods. Molecular cloning techniques that achieve these goals are well known in the art. Numerous cloning methods, as well as in vitro amplification methods, for constructing recombinant nucleic acids are well known to those of ordinary skill in the art.

A natural or synthetic nucleic acid encoding an antibody of the invention is expressed by ligating a nucleic acid encoding an antibody to a promoter (which may be persistent or inducible) and placing the construct in an expression vector. The vector may be suitable for replication and implantation in prokaryotes, eukaryotes, or both. A typical cloning vector comprises a transcriptional and translational terminator, an initial sequence, and a promoter that can be used to regulate the performance of the nucleic acid encoding the antibody. The vector optionally comprises a universal expression cassette comprising at least one independently terminator sequence, a sequence that allows for replication in prokaryotes and eukaryotes, such as a shuttle vector. And screening markers for use in prokaryotic and eukaryotic systems.

In order to increase the high expression of the cloned nucleic acid, it is usually constructed into a plastid, which typically contains a strong promoter for direct transcription, a ribosome binding site for initial translation, and a transcription/translation terminator. . It is also very helpful to include the marker for screening on the DNA vector and then transform it into E. coli . Examples of such markers include genes that are resistant to penicillin, tetracyclines, or chloramphenicol. The expression systems for expressing antibodies are readily available, such as E. coli , Bacillus sp. , and Salmonella E. coli systems.

The antibody gene may also be sub-selected in a performance vector, which may be added to a C-terminus or N-terminus of an antibody (eg, scFv) with a tag (eg, 6 histidine) for purification. Methods for transfecting and expressing genes in mammalian cells are well known in the art. Transduction of the nucleic acid into the cell can comprise, for example, culturing the nucleic acid-containing viral vector with a cell that is the host within the host range of the vector. The cell culture used in the present invention includes cell strains well known in the art or cultured cells derived from tissue or blood samples.

Once the nucleic acid of the antibody has been isolated and cloned, it can be expressed in a variety of different recombinantly engineered cells; wherein the cell line is well known to those of ordinary skill in the art. Examples of such cells include bacteria, yeasts, filamentous fungi, insects (e.g., cells that can be applied to baculovirus vectors), and mammalian cells.

Isolation and purification of the antibodies can be accomplished according to methods well known in the art. For example, the protein can be isolated from the cell lysate, the cell is genetically engineered to be sustainable, and/or induced to express the protein; or by immunoaffinity purification (or precipitation using protein G or A) The protein is isolated from the synthesis reaction mixture; the non-specifically bound material is removed by rinsing, and then the specifically bound antibody is eluted. The isolated antibody can be further purified by dialysis and other methods commonly used for protein purification. In one embodiment, metal chelation and chromatography are used to isolate the antibody. The antibodies of the invention, as described above, may comprise modifications for isolation.

The antibody can be prepared in a substantially pure or isolated form (eg, no other multipeptide is present). The protein may be present in a composition in which the protein is present as an enriched multi-peptide, as compared to other components that may be present (eg, other multi-peptides or other host cell components). The invention may also provide a purified antibody such that the antibody is present in a composition that is substantially free of other proteins, such as less than 90%, typically less than 60%, and more often less than 50% of the composition. It consists of other expressed proteins.

The invention also provides cells useful for producing the antibodies. The cell can be a fused cell or a "fusion tumor" which can produce antibodies (eg, monoclonal antibodies, such as IgG) in vitro.

The present invention also contemplates techniques for making recombinant DNA for the antigen binding region of an antibody molecule, and which does not require the production of a fusion tumor. The DNA is constructed into, for example, bacteria (eg, phage), yeast, insect or mammalian expression systems. An example of a suitable technique is the use of a phage lambda (lambda) vector system with a leader sequence that allows the expression of an antibody (eg, Fab or scFv) to move into the space surrounding the protoplasm (between the bacterial cell membrane and the cell wall) Or may cause the expressed antibodies to be secreted. This quickly produces a large number of functional fragments (eg, scFv) that can be combined with c-Met.

Modification

The invention comprises an antibody and a nucleic acid which can be modified to impart desired properties, such as to facilitate delivery to a specific type of tissue and/or cells in a subject, increase serum half-life, and supplement anticancer activity. Wait. The antibodies provided by the present invention may or may not be modified, and the antibodies provided by the present invention include human antibodies, humanized antibodies, and chimeric antibodies. One method of modifying the antibody is to bind (e.g., link) one or more other elements to the N-terminus and/or C-terminus of the antibody, which may be other proteins, and/or drugs, or carrier molecules.

The antibody modification is ligated to a linker which still retains the desired binding specificity and which, by virtue of the nature of the second molecule being combined, provides the antibody with additional desirable properties. For example, the antibody can bind to a second molecule that helps to increase solubility, storage or other handling characteristics, cell permeability, half-life, reduce immunogenicity, control its release and/or distribution, for example, Target specific cells (eg, neurons, white blood cells, tumor cells, etc.) or the location of cells (eg, solutions, endosomes, mitochondria, etc.), tissue or other body locations (eg, blood, nerve tissue, specific organs, etc.) ). Other examples include that the antibody can be combined with a dye, fluorophore or other detectable label, or reporter molecule for analysis, tracking, and the like. More specifically, the antibody can bind to a second molecule such as a peptide, a multi-peptide, a dye, a fluorophore, a nucleic acid, a saccharide, an anticancer agent, a lipid, and the like ( For example, at the reducing end or the non-reducing end, the second molecule is like a labeled lipid moiety, including N-fatty thiol (such as N-oil sulfhydryl), fatty amines (like ten). Dialkylamine, oleic acid amine and the like).

For example, an antibody can be internalized into a cell, and the antibody or nucleic acid of the invention can be further modified to increase or decrease its efficiency of delivery into the cell. A gene delivery method is also contemplated herein for delivery to a nucleic acid that will express the antibody in a cell. By increasing the association of antibodies with peptides or proteins, the efficiency of antibody uptake by cells (eg, pinocytosis) is increased or decreased. For example, the antibody can be linked to a ligand of the subject receptor or to a macromolecule (like another antibody), which makes it more susceptible to ingestion by the pinocytosis mechanism. When the pinocytosis vesicles are fused to the solution, the carrier linked to the antibody can be released by acid hydrolysis or enzyme activity. To reduce cellular uptake, the linker to the antibody can include a ligand that maintains the antibody on the cell surface, which can be used to control cellular uptake, or in some instances, when lowering a cell type After the absorption of the state, the absorption of another cell type can also be increased.

Other properties of the binding antibody can include reducing the toxicity of the antibody compared to the unbound antibody. Another property is that the conjugated antibody targets a cell or organ of a type (eg, cancer cell or cancer tissue) more efficiently than an unbound antibody.

Other examples include an antibody that binds to one or more molecules that complement, enhance, enhance, or synergize with the antibody. The antibody can be attached to: an anticancer drug, for example, this can be used to deliver the drug to a cancer site for cytotoxic or clearance, for example, an anti-proliferative moiety (eg, a VEGF antagonist, eg, an anti-VEGF antibody), a toxin (eg: doxorubicin, ricin, Pseudomonas aeruginosa exotoxin A and its analogues), radionuclides (eg ⁹⁰ Y, ¹³¹ I, ¹⁷⁷ L, ¹⁰ B for boron neutron capture, and analogues thereof) ), an anticancer agent, and/or an oligonucleotide (eg, siRNA).

A liposome containing an antibody. For example, an antibody can be prepared in a lipid nanoparticle (eg, a liposome) by covalent or non-covalent modification. For example, the antibody can be directly affixed to the surface of the lipid nanoparticle by passing through the Fc region. The antibody can also be covalently attached to the end of the polymer grafted onto the surface of the lipid nanoparticle by a linker. These linked lipid nanoparticles may be referred to herein as "immunolipids".

A gene encoding an antibody of the invention (e.g., S20) can be fused to cysteine at the C-terminus produced. The cysteine fusion protein is specifically coupled to a maleic imine-modified PEG chain located on the outer surface of the liposome via a site-directed conjugation through its C-terminal cysteine. . The immunolipid can carry one or more anticancer agents, such as small molecules, peptides, and/or nucleic acids (e.g., siRNAs), or any of those conventional in the art. For example, the liposome may contain an anticancer drug such as doxorubicin. The antibody acts as a target molecule in an immunolipid to drive the immunolipid to specifically bind to c-Met on the surface of the cancer cell. Methods for preparing and loading lipid nanoparticles, such as liposomes and immunoliposomes, are well known in the art. Please refer to, for example, US Pat. No. 7,749,485 and US Pat.

The antibodies of the invention are optionally modified (e.g., by polyethylene glycolation, hyperglycosylation, and the like) to provide improved pharmacokinetic information. Modifications to enhance the half-life of serum are also of interest to the present invention. When it contains one or more poly(ethylene glycol) (PEG) molecules, the antibody can be "polyethylene glycolated." Reagents and methods for the polyethylene glycolation of proteins are known in the art and can be found in U.S. Patent No. 5,849,860, the disclosure of which is incorporated herein by reference.

When the anti-system is isolated from a source, the protein can be linked to some molecule for purification, such as a member of a specific binding pair, such as biotin (biotin-oval) It is a combination of a single person, a lectin and its analogues. The protein may also be bound (e.g., cured) to a solid support, including but not limited to: polystyrene disks or beads, magnetic beads, test strips, membranes, and the like.

When the antibody is to be detected in the assay, the protein may also contain a detectable label, such as: a radioisotope (eg, ¹²⁵ I, ³⁵ S and its analogs), an enzyme (eg, fluorescent) a chromogenic protein, a phosphogenic protein, a phosphogenic protein, a phosphogenic protein , dyes (such as: fluorescein isothiocyanate, rhodamine, phycoerythrin and its analogues), fluorescent emission metals, such as: ¹⁵² Eu, or other 镧a family element permeable to a metal chelating group (such as EDTA) to attach the metal to the protein; chemical fluorescent compounds such as luminescent amines, isoluminescent amines, acridine salts and the like; bioluminescent compounds Such as: luciferin, fluorescent protein and their analogues. Indirect labels include antibodies specific for the protein, wherein the antibody is detectable by secondary antibodies; and members of specific binding pairs, such as biotin-avidin and the like Things.

Any of the elements described above for modifying the antibody may be linked to the antibody via a linker, such as a flexible linker. In the presence of the linker, the linker molecule is typically of sufficient length to provide for the antibody and a linked carrier between the antibody and the carrier for resilient movement. The linker molecule is typically about 6-50 atomic in length. The linker molecule can also be, for example, an aryl acetylene, a glycol oligomer containing 2-10 single molecule units, a diamine, a dibasic acid, an amino acid, or a combination thereof.

When the linker is a peptide, the linker can be of any suitable length, such as from 1 amino acid (such as Gly) to 20 or more amino acids, from 2 amino acids to 15 Amino acid, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 An amino acid, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Elastomeric linkers include glycine polymer (G) _n , glycine-serine polymers (including, for example, (GS) _n , GGSGS _n (SEQ ID NO: 1), and GGGS _n (SEQ ID NO) 2), wherein n is an integer of at least 1), a glycine-alanine polymer, an alanine-silic acid polymer, and other elastic linkers well known in the art. Glycine and glycine-glycine polymers can be used when relatively unstructured amino acids are desired, and they can be used as a neutral range between the different components. Examples of such flexible linkers include, but are not limited to, GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4), GGSSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7), GSSSG (SEQ ID NO: 8) and analogs thereof. Those of ordinary skill in the art will recognize that any of the foregoing elements are linked to a peptide, and may include all or a portion of a resilient linker such that the linker may comprise a resilient linker and one or more positions. This position gives less elastic structure.

Human genetically engineered antibodies . The antibodies of the invention may be in the form of immunoglobulins, such as human IgG. For example, an antibody of the invention is in the form of an scFV that can be ligated to a human constant region (eg, an Fc region) to produce a human immunoglobulin, such as a complete IgG immunoglobulin.

Fc region . An antibody of the invention that binds to c-Met may contain an Fc region. The Fc region can be any homologue naturally present in a human or other animal (e.g., an immunoglobulin derived from any type or subtype), and the Fc region can optionally be further modified to achieve a different function. When the Fc region is non-human and the CDR and/or FR region is of human origin, the antibody may be referred to as a chimeric antibody. The Fc region can be modified at one or more amino acid residue positions to achieve enhanced utility, such as inducing cellular cytotoxicity, or activating complement activity (eg, C1q binding or complement dependent cytotoxicity), Down regulation of cell surface receptors and the like. A variety of variants of Fc can be used in the antibodies of the invention, as described in, for example, US 7,416,727, US 7,371,826, US 7,335,742, US 7,355,008, US 7,521,542, and US 7,632,497; The disclosure is also hereby incorporated by reference.

combination

The composition provides an antibody, and/or a nucleic acid encoding the antibody, wherein the antibody binds to a cancer cell that exhibits c-Met and can be internalized by a cancer cell. The compositions of the present invention have been found to be useful in the treatment of a subject (e.g., a human) having cancer, and cancer at any stage may be suitable for treatment using the compositions of the present invention.

The compositions provided herein may comprise one, two, or a plurality of different antibodies as a pharmaceutical composition and administered to a mammal in need thereof (e.g., administered to a human).

The compositions described herein may comprise one, two, three, or a plurality of different antibodies (and/or nucleic acids encoding the antibodies) of the invention. For example, the composition may comprise one or more of the following: strains 1, 2, and 3. The composition optionally further comprises an antibody comprising one or more CDRs derived from the antibodies, and/or one or more antibodies comprising a variant or derivative of the antibodies.

Examples of the compositions of the invention may include any of the antibodies disclosed in Table 1. When the composition contains two or more antibodies, each antibody may be specific for the same or different epitopes, or may be specific for the epitope of a different antigen. For example, the composition contains at least one antibody that is specific for the epitope of c-Met, and other antibodies may be specific for other cell surface antigens, such as EGFR. The composition may also contain a dual specificity, a multi-specific antibody, or a nucleic acid encoding the same.

The antibodies of the invention may be used alone, and/or together with other ingredients (eg, to form dual specific or multiple specific antibodies), and/or used in conjunction with other conventional anticancer agents (eg, for the treatment of cancer) Antibody). For example, the composition, such as a liposome, can comprise two or more antibodies, wherein at least one of the antibodies is an antibody of the invention. As described above, the liposome may contain one or more antibodies which are different from the antibodies of the present invention. The liposomes may be of dual specificity, multiple specificity, etc., that is, the liposome is specific to the other antigenic determinant in addition to the specificity of the epitope of the antibody of the present invention.

The combination of the compositions may be provided as a single formulation or as a separate formulation in a set, wherein the separate formulation may contain a single antibody or two antibodies. Separate formulations in the kit can be administered prior to administration or separately for administration.

The pharmaceutical composition may be provided in a pharmaceutically acceptable excipient which may be a solution, such as an aqueous solution, usually a physiological saline solution, or it may be provided in powder form. The compositions may contain other ingredients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium, carbonates, and the like. The composition may contain pharmaceutically acceptable adjuvants to achieve a suitable physiological environment, such as pH adjustment and buffering agents, toxicity modulators and the like, such as sodium acetate, sodium chloride, potassium chloride, Calcium chloride, sodium lactate and the like.

The antibody, if in the form of a pharmaceutically acceptable salt, can be formulated for oral, topical or parenteral administration for use in the methods described below. In certain embodiments, such as when the antibody is administered in a liquid injectable form, the antibody formulation is provided as a ready-to-use dosage form or as a pharmaceutically acceptable carrier and excipient. It can be reconstituted in a stable storage powder or in liquid form.

Compositions of the invention may include a therapeutically effective amount of the antibody, as well as any other compatible ingredients, if desired. "Therapeutically effective amount" is intended to mean a single dosage form, or a series of identical or different antibodies or parts of a composition, which, when administered to a body, is effective in inhibiting the proliferation and/or metastasis of cancer cells in a subject. The amount. The therapeutically effective amount of the antibody can affect cell growth and also includes cooperating with one or more other therapies (eg, immunotherapy, chemotherapy, radiation therapy, etc.), and/or synergistically to inhibit cell growth. As described below, the therapeutically effective amount can be adjusted by visually administering the treatment course and the diagnostic analysis of the patient's condition (for example, using an antibody specific for c-Met to detect the presence or absence of a cell surface antigen group).

Amount and dosage form . The concentration of the antibody may vary from one pharmaceutical preparation to less than about 0.1%, usually or at least about 2%, up to 20% to 50%, or more, and may be The specific mode of choice and the patient's needs are selected by liquid volume, viscosity, and the like. The resulting composition may be in the form of a solution, suspension, tablet, pill, sachet, powder, gel, emulsion, detergent, ointment, aerosol or the like.

Furthermore, it is known that it can act on the desired growth inhibition or immunosuppressive response depending on the anticancer agent or immunosuppressive agent to determine the appropriate dosage and course of administration. These doses include inhibition of cell growth at low doses without significant side effects. The compounds of the present invention provide a wide range of intracellular reactions, such as from partial inhibition to substantially complete inhibition of cell growth, at appropriate dosages, with concomitant administration of a particular compound. The therapeutic dose can be in a single dose mode or in a multiple dose mode (eg, including a progressive dose, and a maintenance dose). As described below, the composition can be administered in combination with other agents, and the dosage and course of administration herein can be varied, depending on the patient's needs.

Combination therapy

There are many different cancer therapies that can be used in combination with the antibodies of the present invention. For example, an agent for chemotherapy or a biological response modifier therapy may be present in a pharmaceutical composition comprising an antibody of the invention, such as an immunolipid. Certain agents that can be used in conjunction with the antibodies are briefly described below.

Chemotherapeutic agents are non-peptidic (eg, non-proteinogenic) compounds that reduce the proliferation of cancer cells and include cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include: alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (eg, vinca) alkaloids, nucleic acids, such as inhibitory nucleic acids (eg, siRNA), And steroid hormones.

Antimetabolites include, for example, folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors.

It can be used as an anti-cancer agent for natural products and their derivatives (such as vinca alkaloids, anti-tumor antibiotics, enzymes, lymphatic hormones, and epipodophyllotoxin). Such as taxanes, such as paclitaxel, and any active taxane derivatives, or pro-drugs.

Other anti-proliferative cytotoxic agents are Navelbene, CPT-11, Anastrazole, Letrazole, Capecitabine, Reloxafine, and ring. Cyclophosphamide, ifosamide, and Droloxafine. Microtubule affecting agents with anti-proliferative activity are also suitable for use. Hormonal modulators and steroids (including synthetic analogs) are also suitable for use.

treatment method

There is provided a method for reducing proliferation of cancer cells by administering an antibody of the invention. Patients with, suspected, or at risk of developing cancer are considered for treatment and diagnosis.

The method involves administering a therapeutically effective amount of an anti-c-Met antibody to a patient in need thereof. Administration of the antibody inhibits cancer cell proliferation, reduces tumor weight, reduces metastasis, and/or improves clinical diagnosis of the patient.

In a mammalian subject, particularly a human, the methods of the invention can be used in a variety of different cancer therapies (including cancer therapies for cancer prevention and diagnosis). The patient has, suspected of having, or has the risk of developing a tumor, all of which are considered for the treatment of the present invention.

In a related embodiment, the subject being treated has cells that exhibit (e.g., overexpress) c-Met. c-Met will be expressed on the surface of cancer cells, and c-Met often exhibits a higher expression on cancer cells than non-cancer cells. This view is very helpful for the treatment methods described herein, as antibodies of the invention can be used to treat cells that exhibit or exhibit c-Met. The antibody can be administered to a patient, for example, when the antigen is not yet detectable, but the treatment is not limited. Antibody therapy can also be initiated before the first sign of the disease is observed, or when the first sign of the disease is likely to occur, or before the diagnosis, or after diagnosis.

For example, cancers that can be inhibited by the methods of the invention include, but are not limited to, cancer, including adenocarcinoma, and especially lung cancer (non-small cells and small cells). Other cancers can also be treated, including those derived from the brain, large intestine, stomach, head and neck, stomach, kidney, liver, and breast.

Type of cancer

The methods described herein can be used to treat or prevent a variety of different cancers, particularly those involving angiogenesis and cancer metastasis. Examples of cancers that can be treated using the methods of the invention are shown below.

Cancers that can be treated by the disclosed methods include, but are not limited to, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a type of skin cancer), squamous cell carcinoma (different tissues), bladder cancer, and metastatic cell carcinoma (including A malignant bladder tumor), bronchial carcinoma, colon cancer, colorectal cancer, gastric cancer, lung cancer, small cell carcinoma including lung and non-small cell carcinoma, adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, Lung adenocarcinoma, sweat gland cancer, sebaceous gland cancer, mastoid carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, breast ductal carcinoma in situ or cholangiocarcinoma, choriocarcinoma, seminoma, Embryonic cancer, Helm's tumor, cervical cancer, uterine cancer, testicular cancer, bone cancer, epithelial cancer, and nasopharyngeal cancer.

Sarcomas that can be treated by the disclosed methods include, but are not limited to, fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic vessels Endothelial sarcoma, synovial membrane, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma and other soft tissue sarcomas.

Solid tumors that can be treated by the disclosed methods include, but are not limited to, glioma, astrocytoma, chorioblastoma, craniopharyngioma, ependymoma, pineal adenoma, hemangioblastoma. , auditory neuroma, oligodendritic cell tumor, meningioma, melanoma, neuroblastoma, and retinoblastoma.

Other cancers that can be treated according to the disclosed methods include: atypical meningioma (brain), islet cell carcinoma (pancreas), thyroid medullary carcinoma (thyroid), stromal tumor (small intestine), hepatoma (liver) , hepatoblastoma (liver), clear cell carcinoma (kidney), and mediastinal nerve fiber tumors.

Examples of more cancers that can be treated by the disclosed methods include, but are not limited to, cancers derived from neuroectoderm and epithelial cells. Examples of cancers derived from neuroectoderm include, but are not limited to, backbone osteosarcoma, spinal cord tumors, brain tumors, supratentorial primitive neuroectodermal tumors of infants, cystic carcinoma, mucinous small tubular and spindle cell carcinoma, renal tumors , mediastinal tumors, gliomas, neuroblastomas, sarcomas of adolescents and young adults. Examples of cancers derived from epithelial cells include, but are not limited to, small cell lung cancer, breast cancer, crystal cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, ovarian cancer, and bronchial epithelial tissue cancer.

Paired with other cancer therapies

Therapeutic administration of the anti-c-Met antibody can be included in one part of the course of treatment, which may or may not be used in conjunction with other standard anti-cancer therapies; including but not limited to: immunotherapy , chemotherapeutic agents and surgery (as mentioned below).

Furthermore, therapeutic administration of an anti-c-Met antibody can also be administered after the subject has been treated with an anti-cancer therapy, such as surgery, radiation therapy, administration of a chemotherapeutic agent, and the like. . Cancer therapy using the fibrillar protein of the present invention can also be used in combination with immunotherapy. In other examples, the fibrillar protein can be combined with one or more chemotherapeutic agents (eg, cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)), and/or Inject with radiation therapy and/or with surgical procedures (eg, before surgical removal of the tumor, or after removal of the tumor). When the fibrillar protein is used in combination with a surgical procedure, the fibrillar protein can be administered before surgical removal of cancer cells, surgical removal of cancer cells, or surgical removal of cancer cells, It can be administered in a systemic manner or in a topical administration at the surgical site. The fibrillar protein may be administered alone or in combination with the foregoing, such as by parenteral administration (eg, by parenteral administration; for example, by intravenous injection), or by topical administration (eg, in a local tumor site). Intratumoral administration (eg, in solid tumors, lymph nodes associated with lymphoma or leukemia), administration to blood vessels supplying solid tumors, etc.).

A variety of different cancer therapies can be used in conjunction with the fibrillar therapy described herein. Such cancer therapies include surgery (eg, surgical removal of cancerous tissue), radiation therapy, bone marrow transplantation, chemotherapy, biological response modifier therapy, and various combinations of the foregoing therapies.

Radiation therapy includes, but is not limited to, X-rays or gamma rays, which can be applied from an externally supplied source, such as a beam of light, or by implantation of a small source of radioactivity.

Chemotherapeutic agents are non-peptidic (eg, non-proteinogenic) compounds that reduce the proliferation of cancer cells and include cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include: alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (eg, periwinkle) alkaloids, and steroid hormones.

Agents useful for reducing cell proliferation are well known and widely used in the art. The agents include alkylating agents such as nitrogen mustard gas, nitrosourea, ethyleneimine derivatives, alkyl sulfonates, and triazenes including, but not limited to, dichloromethyldiethylamine, rings Amides phosphate (CYTOXAN ^(TM)), melphalan (melphalan), dolastatin card curtain (BCNU), dolastatin curtain passage (CCNU), semustine (methylation-CCNU), streptozotocin , chlorourea, uraramustine, clopidogrel, effluranium, chlorambucil, piperac bromide, tritamine, tri-ethyl thiophosphonamide, malilan (Busulfan), up to Carbe, and temamine.

Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU) ), fluorouridine (FudR), 6-thioguanine, 6-巯嘌呤 (6-MP), pentastatin, 5-fluorouracil (5-FU), methotrexate, 10-propynyl-5 , 8-bisazinoyl folate (PDDF, CB3717), 5,8-bis-non-nitrogen tetrahydrofolate (DDATHF), formamidine tetrahydrofolate, fludaben phosphate, dextrosine, and ghita guest.

Suitable natural products and their derivatives (such as vinca alkaloids, anti-tumor antibiotics, enzymes, lymphokines, and epipodophyllotoxin), including but not limited to: Ara-C, paclitaxel (TAXOL) ), European paclitaxel (TAXOTERE) ), deoxyribomycin, mitomycin-C, L-aspartate pronase, azathioprine; buquina; alkaloids, such as vincristine, vinblastine, vinorepine, deacetylate Vinorexine, etc.; podophyllotoxin, such as etoposide, teniposide, etc.; antibiotics, such as anthracycline, daunorubicin hydrochloride (danomycin, bidamycin, daunorubicin), Idarubicin, doxorubicin, anthracycline and morpholinyl derivatives; phenoxazinone bicyclic peptides (such as actinomycin; basic glycopeptides such as bleomycin; glucosides, Such as plicamycin (mitomycin, mithramycin); diamino hydrazine, such as bishydroxyindole; aziridine pyrrolopyridinone, such as mitomycin; giant Cyclic immunosuppressive agents, such as cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, and the like, and analogs thereof.

Other anti-proliferative cytotoxic agents are nalavidin, CPT-11, amnazole, norxazole, capexitabin, rosastene, cyclophosphamide, ibuprofen, and torofloxacin.

Microtubule-influencing agents with anti-proliferative activity are also suitable for use, including but not limited to: isocolchicine (NSC 406042), halikon B (NSC 609395), colchicine (NSC 757), colchicine-derived (eg NSC 33410), Guinea Pig Peptide 10 (NSC 376128), maytansine (NSC 153858), Lissoxin (NSC 332598), paclitaxel (TAXOL) ), TAXOL Derivative, European paclitaxel (TAXOTERE) ), thiocolchicine (NSC 361792), trityl cysterin, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones, including but not limited to: angstrom Paclomycin A, epothilone B, dexmolole; estramustine, nocodazole and the like.

Hormone regulators and steroids (including synthetic analogs) may also be suitable for use, including but not limited to: adrenal corticosteroids, such as prednisone, dimethoprim, etc.; estrogen and progesterone such as: hydroxypregnation Ketone, medroxyprogesterone acetate, megestrol acetate, estradiol, romilifene, tamoxifen, etc.; and adrenal cortical inhibitors, such as amine lutimidin; 17α-ethinylestradiol; diethylstilbestrol, anthrone , fluoromethyl ketone, tacrosterone propionate, azlactone, methylprednisolone, methyl ketone, prednisolone, triamcinolone, ketene estradiol, hydroxyprogesterone, amine ubmet, ermus Tym, medroxyprogesterone acetate, leuprolide, drogenil, faremis, and ZOLADEX . Estrogen can stimulate proliferation and differentiation, so compounds that bind to the estrogen receptor can be used to block the activity of estrogen. Corticosteroids can be used to inhibit proliferation of T cells.

Other chemotherapeutic agents include metal complexes such as cis-DDP, carboplatin, etc.; urea, such as hydroxyurea; and hydrazine, such as N-formamidine; eppylopoxin; topoisomerase inhibitors ; procarbazine; mitoxantrone; formazan tetrahydrofolate; Other anti-proliferative agents of the present invention include immunosuppressive agents such as mycophenolic acid, thalidomide, deoxyspirin, azaporine, leflunomide, imidazoribine, spirocycloalkane (SKF) 105685); IRESSA (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline)) and the like.

"Taxanes" include paclitaxel, as well as any active taxane derivatives, or prodrugs. "Paclitaxel" (which should be understood herein to include analogs, formulations, and derivatives such as, for example, docetaxel, paclitaxel (TAXOL), TAXOTERE (polytaxol), 10-off The acetamidine-paclitaxel analogs as well as the 3'N-desphenylhydrazin-3'Nt-butoxycarbonyl paclitaxel analogs can be prepared in the art by techniques well known to those skilled in the art.

Paclitaxel is understood to be not only a common chemically available form of paclitaxel, but also analogs and derivatives thereof (TAXOTERETM docetaxel as described above), as well as paclitaxel conjugates (eg paclitaxel-PEG, paclitaxel-grape) Glycan, or paclitaxel-xylose).

In the treatment of a part of patients according to the method of the present invention, it is necessary to use a high dose therapy and a rescue agent for non-malignant cells. In such therapies, any agent that can be used to rescue non-malignant cells can be used, such as Citrovorum factor, folate derivative, or leucovorin. Such rescue agents are well known to those of ordinary skill in the art. Rescue agents include such rescue agents that do not interfere with the compounds of the invention to modulate cellular function.

Route of investment

The anti-c-Met antibody can be administered to different parts of the body by various means, including intratumor, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (eg, topical). , transmucosal, intraperitoneal, intra-arterial, and rectal administration. Other suitable routes include oral, buccal, nasal, nasopharyngeal, parenteral, enteral, transgastric, topical, transdermal, subcutaneous, intramuscular, The composition is administered by intralesional injection into a tumor, solid, powder, liquid, or mist to inject the tumor, or to inject into the tumor, intravenous infusion, and arterial infusion. The mode of administration can be topical or systemic, with or without the addition of excipients. Administration can also be accomplished by slow release on or near the tumor site of the patient.

It will be apparent to those of ordinary skill in the art that a variety of different methods suitable for administering the formulations of the present invention can be used to administer a patient or host, such as a patient in need thereof, and although more than one path can be used A particular recipe is cast, but there is still a specific delivery path that makes it more immediate and more efficient than another path.

The term "therapeutically effective amount" refers to the amount of effect that can be exerted at a reasonable benefit/risk ratio when applied to medical treatment. The effective amount will vary depending on factors such as the disease or condition being treated, the administration of the particular subject, the size of the patient, or the severity of the disease or condition. An effective amount of the particular compound can be determined by one of ordinary skill in the art based on experience and without the need for degree testing.

According to an illustrative example, the protein can be administered as part of a composition, as will be explained in more detail below. The compositions may be in various forms, including powders, milks, gels, ointments, ointments, solutions, tablets, sachets, sprays, and patches. The carrier and carrier can be used to deliver the composition to a patient. The carriers include solvent, diluent and dispersant. The carrier is biocompatible, pharmaceutically acceptable, and does not alter the therapeutic properties of the fibrillar protein. Excipients, adjuvants and other ingredients may also be included in the composition.

dose

In this method, an effective amount of an anti-c-Met antibody is administered to a patient in need thereof. The dose to be administered depends on the intended purpose, the health and physiological condition of the individual being treated, the age and type of the individual being treated (eg, human, non-human primate, primate, etc.) The degree, formulation of the anti-c-Met antibody composition, the clinician's assessment of the medical condition, and other relevant factors will vary. It is expected that the effective amount will be very broad and can be determined by routine experimentation. For example, an effective amount of an anti-c-Met antibody to inhibit cancer metastasis cannot be more than an amount that causes irreversible toxicity to a patient (eg, a maximum tolerated dose). In other instances, the effective amount is about or nearly slightly below the limit of toxicity, but is still immunologically effective, or as low as the limit dose.

The individual dose is usually not lower than the amount that allows the patient to produce a measurable effect, and the dose can be based on the pharmacokinetics and absorption, distribution, metabolism, and excretion (ADME) of by-products of the anti-c-Met antibody. Pharmacology, and depending on the configuration of the composition in the patient, is determined. This includes consideration of the route of administration and dosage, which can be adjusted for topical application (where it is intended for topical utility, where it can be applied directly), and enteral application (when it is part of the digestive tract) , through the digestive tract for systemic or local utility), non-orbital applications (by non-digestive pathways to provide systemic or local utility). For example, administration of an anti-c-Met antibody can typically be by injection, and often intravenous, intramuscular, intratumor, or a combination thereof.

The anti-c-Met antibody can be administered by injection or local injection, for example, at a rate of from about 50 mg/h to about 400 mg/h, and also includes from about 75 mg/h to about 375 mg/h. From 100 mg/h to about 350 mg/h, from about 150 mg/h to about 350 mg/h, from about 200 mg/h to about 300 mg/h, from about 225 mg/h to about 275 mg/h. Exemplary injection rates can be used to achieve a desired therapeutic dose, for example, from about 0.5 mg/m ² /day to about 10 mg/m ² /day, including from about 1 mg/m ² /day to about 9 mg/m ² / Day, about 2 mg/m ² /day to about 8 mg/m ² /day, about 3 mg/m ² /day to about 7 mg/m ² /day, about 4 mg/m ² /day to about 6 mg /m ² /day, about 4.5 mg/m ² /day to about 5.5 mg/m ² /day. Repeated administration (eg, injection) in a desired cycle, such as a period of about 1 day to 5 days, or every few days (eg, 5 days, more than about 1 month, about 2 months, etc.) Repeated administration. It can also be administered before, during, and after other treatments (such as surgery to remove cancer cells). The anti-c-Met antibody can also be administered as part of a combination therapy wherein at least one of immunotherapy, cancer chemotherapy, or radiation therapy is administered to the patient (described in more detail below).

The distribution of anti-c-Met antibodies in a patient and their corresponding biological activity can typically be measured by a fragment of an anti-c-Met antibody present on the subject of interest. For example, when an anti-c-Met antibody is administered, the antibody can be accumulated together with a glycoconjugate or other biological target, and the substance is concentrated in cancer cells and cancer tissues. Thus, when an anti-c-Met antibody is administered, such that it accumulates within the desired amount over a period of time, this will be part of the dosing regimen for lower individual dosages. This also means that, for example, since the dose of the anti-c-Met antibody is cleared in vivo, the concentration of the antibody can be lower than the effective concentration calculated in the in vitro test (eg, effective in vitro). The amount is about mM, while the concentration in vivo is lower than mM).

As an example, an effective dose or amount of program administration, by an anti-c-Met antibody administered for inhibition of cell migration measured by IC _50. "IC ₅₀ " means a drug concentration that is used in vitro to achieve a 50% inhibition rate. Alternatively, the effective amount of antibody concentration by an anti-c-Met EC ₅₀ of given measured. "EC ₅₀ " is intended to be used in vivo to achieve a blood concentration of 50% of maximum utility. In related embodiments, the dosage may decide based on the ED ₅₀ (effective dose).

In general, the effective amount of the anti-c-Met antibody of the present invention generally does not exceed the calculated IC ₅₀ 200X. Typically, the amount of administered anti-c-Met antibodies based on the calculated sum is lower than the IC ₅₀ of about 200X, 150X lower by about, about IOOX low, and in many embodiments, the low of about 75X, a low of about 60X, 50X, 45X 40X, 35X, 30X, 25X, 20X, 15X, 10X, and even less than the calculated IC _{50 of} about 8X or 2X. In one specific embodiment, the effective amount is calculated as the IC ₅₀ of 1X to 50X, and the calculated IC sometimes about ₅₀ 2X to 40X, 30X to about 3X, or 4X to 20X. In other embodiments, the effective amount of the same system of calculation of IC _50, and in certain embodiments, the amount of calculation of the IC ₅₀ of the effective amount of the above system.

An effective amount may not exceed the calculated EC ₅₀ 100X. For example, an anti-c-Met antibody is to EC ₅₀ of less than about IOOX calculated, less than about 50X, less than about 40X, 35X, 30X, or 25X, and in many embodiments about 2OX low, a low of about 15X And an amount of about 10X, 9X, 8X, 7X, 6X, 5X, 4X, 3X, 2X, or 1X which is lower than the calculated EC ₅₀ is administered. The effective amount is about 1X to 30X of the calculated EC ₅₀ , sometimes about 1X to 20X, or about 1X to 10X. It may be the same or higher than the EC ₅₀ calculated with the effective amount of the calculated EC _50. The EC ₅₀ can be moved by the cell in vitro / inhibition rate of infiltration is calculated. The steps can be carried out by methods well known in the art or as described in the examples below.

The effective amount of the dosage and/or dosage regimen can be determined empirically by trial, from safe, large scale, and dose range trials, individual physician patient relationships, and in vitro and in vivo assays.

diagnosis method

The invention provides methods for detecting c-Met (e.g., full length or fragment) in a biological sample of a patient, or in a sample isolated from a patient. This method is very helpful for the purposes of diagnosis and prognosis. The method generally involves contacting a sample containing the cells with an antibody of the invention; and detecting the condition of binding of the antibody to the cells within the sample. The cell may be in vitro, meaning that the cell line is present in a biological sample from a patient suspected of having cancer cells, a patient undergoing cancer treatment, or a patient to test for susceptibility to treatment. The cell may be in vivo, for example, the cell line is present in a patient suspected of having cancer cells, a patient undergoing cancer treatment, or an individual for testing a patient susceptible to treatment.

The anti-system can be used in biological samples to detect cells that express c-Met by immunodiagnostic techniques derived from an individual with or suspected of having cancer cells. These diagnostic methods (described in more detail below) are useful for identifying patients who can be treated and/or monitoring the response to treatment.

Suitable immunodiagnostic techniques include, but are not necessarily limited to, in vitro and in vivo (imaging) methods. For example, an anti-c-Met antibody can be labeled with a detectable label to administer a suspected cancer-bearing individual (characterized by cell surface expression of c-Met) and available in the field. An imaging method that detects the binding of the labeled antibody that can be detected.

As used herein, the term "in vivo imaging" refers to a method for detecting the presence of an antibody (such as a selected strain 21 labeled by a detectable label) in a whole body or living mammal. Optically detectable proteins, such as fluorescent antibodies, and fluorescently conjugated antibodies, can be visualized in vivo. In vivo visualization of fluorescent proteins in living animals has been described, for example, in Hoffman, Cell Death and Differentiation 2002, 9:786-789. In vivo imaging may also provide images of 2D and 3D mammals. Charge-coupled device cameras, CMOS, or 3D tomographic images can be used for in vivo imaging. For example, Burdette JE Journal of Mol. Endocrin ., 40: 253-261, 2008, mentions the use of computed tomography, magnetic resonance imaging, ultrasound imaging, positron tomography, single photon emission computed tomography (SPECT) and the like. The information obtained by the above-mentioned many in vivo imaging methods can be used to know the information of cancer cells in patients.

When the method is in vitro, the biological sample can be a variety of samples that may contain cancer cells, including but not limited to blood samples (including whole blood, serum, etc.), tissues, whole cells (eg, intact cells), and tissues. Or cell extracts. For example, the test method can be used to detect c-Met located on a living cell or in a tissue sample.

In particular, detection can be performed on the outer surface of the living cells. For example, the tissue sample can be fixed (eg, treated with formaldehyde), and may be embedded in a support (eg, paraffin), or frozen non-fixed tissue.

The test can be carried out in a variety of different forms, such as competitive, direct reaction, or sandwich type testing. Examples of such assays include Western blotting, agglutination assays; enzyme-labeled and vehicle immunoassays, such as enzyme-linked immunosorbent assays (ELISAs); biotin/glycoprotein type assays; Immunoassay; immunoelectrophoresis; immunoprecipitation and its analogs. The reaction generally involves conjugating a detectable tag to the antibody. Labels include such fluorescent, chemiluminescent, radioactive, enzymatic and/or dye molecules, or other antibodies that can be used to detect antigens and antibodies in a sample, or related reactions. Methods.

When a solid support is used, the solid support will typically react with a solid phase component under suitable binding conditions for the antibody to be sufficiently cured on the support. Occasionally, the antibody may be first bound to a protein having a preferred binding property to enhance its curing on the support; or the antibody may be cured on the support without significant loss of the antibody. Binding activity or specificity. Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumin, including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroid gland Globulin, ovalbumin, and other proteins known to those of ordinary skill in the art. Other molecules that can be used to bind antibodies to the support include polysaccharides, polylactic acids, polyglycolic acids, polyamino acids, amino acid copolymers, and the like, provided that the molecules used to cure the antibody do not affect the antibody. Specific binding ability to antigen. Such molecules and methods for coupling such molecules to antibodies are well known to those of ordinary skill in the art.

An ELISA method can be used in which the pores of the microtiter plate are coated with the antibody. A biological sample containing or suspected of containing c-Met is added to the coated pores. After incubation for a period of time sufficient for antibody binding, the plate is washed to remove unbound molecules and the labeled secondary binding molecules that can be detected are added. The secondary binding molecule can react with any captured antigen, wash the disk, and use conventional methods in the art to detect the presence or absence of the secondary binding molecule.

If it is desired to know whether the binding of the antibody of the present invention to the c-Met of the biological sample is detected, it can be detected by using a secondary binder comprising an antibody which can directly bind to the antibody. Receptor. For example, a wide variety of anti-bovine immunoglobulin (Ig) molecules known in the art can be conjugated to a detectable enzyme tag by methods well known in the art, such as spicy Root peroxidase, alkaline phosphatase, or urease. A suitable enzyme is then applied to generate a detectable signal. In other related embodiments, conventional methods in the art can be used to implement competitive ELISA techniques.

This assay can also be carried out in solution, ie, in a precipitating environment, the antibody forms a complex with the antigen. An antibody-coated particle that, in a suitable binding environment, can be contacted with a biological sample suspected of containing the antigen of interest to form a particle-antibody-antigen-mismatched aggregate, which can be agglomerated using rinsing and/or centrifugation. The material precipitated and separated from the sample. The reaction mixture can be analyzed by any standard method to determine whether an antibody-antigen complex is present; such standard methods are as described above for immunodiagnostic methods.

Alternatively, other diagnostic techniques can be used to analyze cellular uptake in living cells to correctly identify cancer cells. Since the antibody is specifically internalized by the c-Met cell, the cell can be used to internalize the antibody, and other antibodies that are not internalized will be washed out (eg, pickled). The internalized antibody can be detected by labeling it in a cell (eg, FACS, spectrometer, radioisotope counter, etc.).

The diagnostic assays described herein can be used to determine if a patient has cancer and whether it requires more or less antibody-based treatment and to monitor the progress of the patient's treatment. It can also be used to analyze the process of other combination therapies. Therefore, the diagnostic method allows the physician to know how to choose the therapy, as well as the course of treatment.

The aforementioned test agents, as well as the antibodies comprising the invention, can be provided in kits, with applicable instructions, and other necessary agents to perform the aforementioned immunoassays. The kit may also contain appropriate labels and other attached agents and materials (eg, wash buffers and the like) depending on the particular immunoassay used. Standard immunoassays, such as those described above, can be performed using such kits.

Sets and systems

The present invention also provides kits and systems that can be used to carry out the foregoing methods. For example, such kits and systems can include one or more of the compositions described herein, such as an anti-c-Met antibody (eg, a selection strain 20 or a selection strain 21), a nucleic acid encoding the antibody (especially A nucleic acid encoding a CDR of a heavy chain and/or a light chain of any of the foregoing antibodies, or a cell containing the antibody or nucleic acid. Other optional components of the kit include: a buffer or the like for administration of the antibody, and/or for performing a diagnostic test. The recombinant nucleic acid in the kit may also have a restriction cleavage site, a multiple cloning site, a primer position, etc., to facilitate ligation to the constant region of the nucleic acid. The different components of the kit may be presented in separate containers, or some compatible ingredients may be pre-mixed in a single container (if needed).

Kits and systems for performing such methods can include one or more pharmaceutical formulations comprising the antibody compositions described herein. For example, the kits can include a single pharmaceutical composition in one or more unit doses. The kit can also include two or more separate pharmaceutical compositions.

In addition to the above ingredients, the kit may further include instructions to perform such methods. The instructions may be present in the kit in different forms and may be present in one or more of the kits or on the kit. One of the descriptions may be in the form of attaching the printed information to a suitable medium or substrate, such as: one or more sheets of paper with information printed on the package or on the package, in the package insert. . Or other means can be a computer readable medium, such as a floppy disk, a CD, etc., and the required information is recorded on the medium. Or other means may be a web address, which can be used to know the information located at the far end via the network. Any convenient way can be presented in the kit.

The invention provides a kit for treating a host having a cell proliferative disorder. The kit includes a pharmaceutical composition comprising an antibody specific for c-Met, and a specification which can be used to illustrate the effective use of the pharmaceutical composition for treating a host having cancer to inhibit the disease The growth of cancer cells in the body. Such instructions include not only appropriate operational characteristics, dosing regimens, and methods of administration and the like, but may further include instructions on how to selectively screen for patients with diseases associated with c-Met. This view may facilitate the use of the user of the kit to assess the likely reactivity of the patient when treated with the antibody of the invention, including the duration of treatment and duration of treatment associated with the type and stage of development of the cancer. Thus, in other embodiments, the kit may further comprise an antibody or other agent for detecting the c-Met epitope on the extracellularly accessible surface of the cancer cell. In another embodiment, the kit comprises an antibody comprising a conjugated detectable tag, such as a fluorophore.

As used herein, the term "system" refers to an antibody as described herein, and a collection of one or more second therapeutic agents, which are present in a single or different composition, which are used together to effect the practice. The purpose of the method. For example, according to the present invention, a separately obtained antibody specific for c-Met, which is placed together with a chemotherapeutic agent, and used together in a patient, is a system.

The following examples are provided to provide a general disclosure of the present invention, and are intended to be illustrative of the invention, and are not intended to limit the scope of the invention.

experimental method

Cell line and culture

PC3 (prostate cancer), human lung cancer cell lines including H1993, H460, H441, A549 and CL1-5, which can be used in an environment containing 5% CO ₂ humidity at 37 ° C in RPMI 1640 containing 10% FBS (Invitrogen) Growing in. SAS (oral cancer), HCT116 (colorectal cancer), Mahlavu (liver cancer), NPC-TW04 (nasopharyngeal carcinoma) (Lee et al. (2004) Cancer Res 64: 8002-8008), PaCa (pancreatic cancer), U2OS (Osteosarcoma) and 293T lines can be grown on DMEM containing 10% FBS. A498 (kidney malignancy) can be cultured on MEM. MDA-MB231 (breast cancer) can be cultured in F12 medium with 50% DMEM in a 10% CO ₂ environment. CL1-5 was established by Chu et al. (1997) Am J Respir Cell Mol Biol 17: 353-360. Mahlavu was presented by Dr. Hsiao M (GRC, Academia Sinica, Taiwan). Other cell lines were obtained from the American Type Culture Collection. Human umbilical vein endothelial cells (HUVECs), and human normal nasal mucosa (NNM) cells have been described in previous studies (Lee et al. (2007) Cancer Res 67: 10958-10965; Lee et al. 2004) Cancer Res 64: 8002-8008).

Construct a phage library that expresses human native scFv

Briefly, cDNA was synthesized from a mixture of mRNA from seven individual samples of human spleen cell tissue using an oligo dT primer and reverse transcriptase (Invitrogen) Superscript III. Transgenic lines of V _H and V _L by PCR, using a polymerase PfuUltra (EMD Biosciences), and specific primers (Marks et al., 1991) increase. The PCR product can be isolated from the gelatin using a nucleic acid purification kit (Qiagene) and purified. The V _H and V _L coding region of the gene, can be by a combination of a DNA fragment encoding the (GGGGS) ₃ amino acid residues of which by the PCR-based, and specific primers containing restriction enzyme cleavage, the cleavage sites are located 5' ( SfiI ) and 3' ( NotI ) ends are reached. The combined product was digested with restriction enzymes SfiI and NotI (NEB), and then ligated into pCANTAB-5E phage vector (GE Healthcare). The human pCANTAB-5E vector containing the scFv gene was electroporated into TG1 E. coli competent cells. After electroporation, TG1 E. coli cells were recovered and cultured on 2YT medium (BD) containing 100 μg/ml penicillin and 2% glucose (2YT-AG). Next, the TG1 cells are rescued with M13KO7 phage, and then phage particles are produced in the medium, which will express scFv.

Phage display of phage expressing anti-c-Met scFv by phage library biopanning

The phage which can express the specific anti-c-Met scFv can be screened by the protein G dynabeads (Invitrogen). The c-Met-Fc recombinant protein (R&D) was incubated with protein G dynabeads for 1 hour at room temperature (RT). The scFv phage library (2 x ¹⁰⁹ species) was first non-specifically bound on protein G dynabeads, and then incubated with c-Met-Fc-cured dynabead for 1 hour at 4 °C. The cells were then washed 4 times with PBST to remove unbound phage. Further, Escherichia coli TG1 cells were used at 37 ° C for 30 minutes to recover phage which binds to c-Met-Fc. A portion of the phage-infected E. coli cells were serially diluted for quantification, and the rest were rescued by M13KO7 helper phage (NEB). After quantifying the price of the rescued phage, the next round of biopanning can be performed. In the fourth and fifth biopanning, phage strains were randomly selected for culture and subjected to ELISA screening.

The selected phage strain was evaluated for binding specificity for 293T cells that would overexpress c-Met.

In immunofluorescence staining analysis, 293T cells were cultured on coverslips to 80% cell density. The pEF-c-Met expression vector was then transfected using Lipofectamin 2000 (Invitrogen) and according to the manufacturer's protocol. After 48 hours of transfection, the cells were incubated with the selected anti-c-Met phage, or control phage, at a titer of 1 x 10 ¹⁰ phage for 30 minutes at 4 °C. The cells were then washed twice with PBS, fixed in 4% paraformaldehyde, and then densified with 0.1% Triton X-100 and blocked with 3% BSA. The cells were probed with rabbit anti-myc antibody (Sigma-Aldrich), and mouse anti-M13 phage antibody, followed by Rhodamine-labeled goat anti-rabbit IgG, and FITC-labeled anti-mouse IgG, respectively. The nucleus was stained with DAPI. The fluorescent images were then photographed by an inverted fluorescent microscope (Axiovert 200M, Zeiss).

For flow cytometry analysis, 293T cells were cultured to 80% cell density and transfected with the pEF-c-Met expression vector in the manner described above. After 48 hours, the transfected cells were harvested with 0.05% trypsin-EDTA, and then incubated with the selected phage, or control phage, at a titer of 1 x 10 ¹⁰ phage in FACS buffer ( The cells were incubated at 4 ° C for 1 hour in PBS containing 1% fetal bovine serum. After washing the cells with FACS buffer, the cells were incubated with mouse anti-M13 phage antibody at 4 ° C for 1 hour, and then conjugated with R-phycoerythrin for goat anti-mouse IgG (Jackson ImmunoReSearch), incubated at 4 ° C for 30 minutes. FACSCanto II (BD) was used for analysis, and the fluorescence intensity of the divergence was measured by FACSDiva software (BD) to quantify its binding affinity.

Performance and purification of soluble scFv.

Anti-c-Met scFv phage selection strains 1, 20 and 21 were respectively infected with Escherichia coli HB2151 strain, followed by preparation of bacterial protoplast clearance extract. Soluble scFv was purified from the protoplast clearance extract using a Protein L agarose column (Thermo Scientific) according to the manufacturer's instructions. The purified scFvs were further dialyzed with PBS, followed by reduced SDS-PAGE, and then by detection of anti-E-labeled antibodies (GE Healthcare) for protein staining (Coomassie blue staining) and Western blot analysis. analysis.

A mammalian expression vector encoding a human c-Met gene is constructed.

All human c-Met cDNA (NM_001127500.1) was synthesized from total RNA of HepG2 cells by Superscript III reverse transcriptase using a specific primer, and then used as a template for PCR. The full-length c-Met-encoding DNA was amplified by PCR using PfuUltra enzyme and ligated in frame with the myc-tagged epitope EF vector (Invitrogen) in the correct frame reading format (invitrogen). Generate pEF-c-Met-myc. DNA fragments encoding c-Met amino acid residues 1 to 932, and 1 to 567 were amplified by PCR and then constructed into pEF vectors to prepare pEF-c-MetF ₉₃₂ and pEF-c, respectively. -MetF ₅₆₇ . The coding region of the human IgG ₁ Fc was obtained from a cDNA library of human spleen cells, and then ligated into the carboxy terminus of c-MetF ₉₃₂ and c-MetF ₅₆₇ in a correct reading frame to generate separately. pEF-c-MetF _932- Fc and pEF-c-MetF _567- Fc.

Production and purification of recombinant protein c-Met truncated

Was to Lipofectamin 2000, the pEF-c-MetF ₉₃₂ -Fc or the c-MetF ₅₆₇ -Fc were transiently transfected into 293T cells, 293T cells can be cultured from the supernatant prepared and c-MetF ₉₃₂ -Fc c -MetF ₅₆₇ -Fc fusion protein. After 72 hours of transfection, the filtered supernatant was passed through a 1 ml Protein G vegetable gel column (GE healthcare). After washing with PBS, the bound protein was eluted with 0.1 M glycine-HCl pH 2.8, followed by neutralization with 1 M Tris-HCl pH 9.1. The eluate was dialyzed extensively with PBS, followed by concentration with an Amicon Ultra-4 centrifugal filter (cutoff 10 kDa; Millipore).

Internalization of anti-c-Met scFv by conjugated focal microscope

H1993 cells, H1993, H460, and c-Met-knocked were seeded on coverslips and grown to a cell density of 80%. The cells were incubated with anti-c-Met scFv S1 or S20 for 30 minutes at 4 ° C and 37 ° C. After rinsing twice with PBS, the cells were fixed with 4% paraformaldehyde and blocked by the addition of 3% BSA. The cells were then stained with anti-Flag antibody (Sigma-Aldrich), followed by FITC-labeled goat anti-mouse IgG (Jackson ImmunoResearch), and DAPI (Invitrogen). All fluorescence images were taken by a conjugated focus microscope (TCS-SP5, Leica).

Construction of prokaryotic expression vector to produce anti-c-Met scFv20-cys (Ms20)

pCANTAB-5E was digested with NotI-EcoRI enzyme to remove the E-tag and pIII DNA fragments, and then passed through the NotI-EcoRI site to insert a gene encoding FLAG tag, six histidine, and cysteine residues. DNA fragment to obtain prokaryotic expression vector pFHC. The anti-c-Met S20 gene was digested from pCANTAB-5E-S20 and passed through the NcoI-NotI site to bind to the pFHC vector. The constructed vector was transformed into E. coli BL21 (Novagen) to express scFv containing six histidine acids and cysteine at the carboxyl terminus.

Performance and purification of anti-c-Met scFv20-cys (Ms20)

Single colony of Escherichia coli BL21 was inoculated into Terrific broth (TB; MD Bio, Taiwan) (TB-A) containing 100 μg/ml penicillin, and cultured at 30 ° C overnight. The overnight culture will be cultured in a fresh 1/50 dilution volume in another fresh 2.5 liter TB-A and cultured at 30 °C until its OD600 reaches 0.5. Next, induction was carried out by adding a final concentration of 0.4 mM IPTG and dissolving 0.4 M sucrose directly in TB-A. The culture method was incubated at 30 ° C and 250 rpm and continuously cultured for 16 hours.

The supernatant was removed by centrifugation at 20,000 x g for 30 minutes, followed by centrifugation of the bacterial mass into cold 200 ml of TES buffer (10 mM Tris, 20% sucrose, and 1 mM EDTA pH 8.0; EMD Biosciences). Resuspend, then incubated at 4 ° C with gentle agitation for 1 hour. The osmotic shock fraction was collected by centrifugation for 30 minutes at a rate of 22,000 x g. The protoplast surrounding extract was obtained by incubating the pellet in ice-cold 5 mM MgSO ₄ with gentle agitation for 1 hour.

The osmotic shock zone and the protoplast surrounding gap extract were mixed to recover the most MS20, then NaCl was added to the mixed sample at a final concentration of 0.5 M, and imidazole at a final concentration of 0.5 mM. A 5 ml Ni ⁺ -NTA gel column (GE Healthcare) was equilibrated with 15 ml of binding buffer (20 mM Tris, 0.5 M NaCl and 5 mM imidazole pH 7.9) and the sample was added. The bound Ms20 was then eluted with elution buffer (20 mM Tris, 0.5 M NaCl and 1 M imidazole pH 7.9). The eluate was thoroughly dialyzed twice with PBS at 4 °C. After dialysis, the sample was again purified by Protein A Agaric Column (2 ml) (GE Healthcare). The repurified Ms20 was dialyzed twice with HEPES buffer (20 mM HEPES, 150 mM NaCl and 1 mM EDTA, pH 7.0), followed by concentration with a 10 kDa cut-off tube (Amicon Ultra, Millipore).

Measuring binding kinetics

The affinity and kinetics of the anti-c-Met scFv were measured by SRP by BIAcore X (GE healthcare). 30 μg/ml of c-MetF _932- Fc protein was coupled to EDC- and NHS-activated CM5 sensing wafers in a BIAcore flowcell, followed by blocking with ethanolamine according to the manufacturer's protocol. The combined phase and separated phase were monitored at 3 and 5 minutes, respectively, using a scFv concentration of 5 to 200 nM at a continuous flow rate of 30 μl/min. Regeneration was carried out by injecting regeneration buffer (0.2 M NaCl, 10 mM glycine, pH 2.7). Binding constants were determined using BIAevaluation software (GE healthcare) to allow the sensorgram to fully conform to the sample 1:1 relationship pattern.

Competitive combination test

To monitor the inhibitory effect of scFv, we coated the c-MetF _932- Fc protein on a 96-well plate and blocked it with 3% BSA (dissolved in PBS) for non-specific binding. 1 nM human HGF (R&D), and various concentrations of anti-c-Met scFv or normal mouse IgG were then added to the wells. Goat anti-human HGF (R&D) was used to determine the amount of HGF bound to c-Met, followed by HRP-labeled mouse anti-goat IgG (Jackson ImmunoResearch) and H ₂ O ₂ plus OPD receptor (Sigma). to cultivate. The absorbance was measured at a wavelength of 490 nm using a microdisk analyzer. The inhibition rate was calculated by the following formula: [(HGF has no absorbance combined with competitors) - (HGF absorbs light combined with competitors)] / (HGF has no absorbance combined with competitors) x 100%.

Construction of Ms20 Targeted Liposomal Doxorubicin (Ms20-LD)

First, it is confirmed that the purified Ms20 has a reduced thiol group for conjugate binding, and the purified Ms20 is 2 mM TCEP (paraxyl (2-carbonylethyl) phosphine; Sigma under N ₂ atmosphere at room temperature; -Aldrich) treatment for 2 hours to reduce the intermolecular disulfide bond of Ms20. The reduced Ms20 was eluted with a HiTrap G-25 column (GE healthcare) in HEPES buffer for desalting. Incorporation of maleic imine-carbonyl polyethylene glycol (M _r 2,000)-derived distearyl phospholipid ethanolamine (maleimide-PEG-DSPE; NOF, Japan) into pegylated liposome The manner of the body doxorubicin (Lee et al., 2004; Lo et al., 2008) will be explained below. The maleic imine-PEG-DSPE was dissolved in HEPES buffer and added to 0.5 mol% of the liposome phospholipids in LD, followed by the mixture at 60 ° C with gentle agitation for 1 hour. to cultivate. The reduced Ms20 was then combined with the maleic imine-PEG-DSPE-embedded LD at 4 ° C overnight to form about 60 molecules of Ms20 in one liposome. After using 2-mercaptoethanol (2 mM final concentration) to deactivate all unreacted maleimine groups, we removed the release by gel filtration chromatography using Sepharose 4B (GE healthcare). Free drug, unbound scFv, and unintegrated conjugate. The concentration of doxorubicin in the eluate can be measured using a spectrofluorometer (Spectra Max M5, Molecular Devices) at λ _Ex/Em = 485/590 nm. The Ms20-LD segment can be separated by reduced SDS-PAGE followed by silver nitrate staining to assess the efficiency of binding.

Identification of Ms20-LD binding to human lung cancer cells

The cells were cultured in 12-well plates to a cell density of 90%. The cells on the plate were incubated with serially diluted LD or Ms20-LD (0.625-10 μg/ml doxorubicin) for 1 hour at 4 °C. After the incubation, the cells were washed with PBS and dissolved in 200 μl of 1% Triton X-100. 300 μl of IPA (0.75 N HCl in isopropanol) was added to the solution and shaken for 30 minutes to extract doxorubicin. After centrifuging the lysate at 12,000 rpm for 5 minutes, the doxorubicin in the supernatant was measured by a spectrofluorometer at λ _Ex/Em = 485/590 nm. The concentration of the doxorubicin was calculated by interpolation using a standard curve.

Ms20-LD is absorbed by human cancer cells

Tumor cells were cultured in a 12-well plate to a cell density of 90%, and 2.5 μg/ml of Ms20-LD or LD in complete medium was added, followed by incubation at 37 ° C for the indicated time. After the cells were washed with PBS, they were subjected to 0.1 M glycine pH 2.8 for 10 minutes to remove Ms20-LD and LD on the cell surface. The amount of doxorubicin absorbed by the cells can be detected by the aforementioned means.

Analysis of Ms20-LD in cell uptake by conjugated focal microscope

H1993 cells were cultured on coverslips to subconfluency. The cells were cultured at 37 ° C at different time points in complete medium containing 2.5 μg/ml of Ms20-LD or LD. After washing twice with PBS, the cells were fixed with 4% paraformaldehyde and blocked by the addition of 3% BSA. The cell membrane and nucleus were stained with Alexa Fluor 647-conjugated wheat germ agglutinin (Invitrogen) and DAPI (Invitrogen), respectively. Intracellular doxorubicin can detect fluorescence at λ _Ex/Em = 485/590 nm. All fluorescent images can be photographed with a laser scanning conjugated focus microscope (TCS-SP5, Leica) and analyzed with LAS AF software (Leica).

Cell survival test

The cells were seeded in 96-well plates at a density of 3000 cells per well and treated with different concentrations (0-20 μg/ml) of MS20-LD or LD in medium (10% FBS) at 37 °C. 24 hours. After removing too much of the agent, the cells were washed once with PBS, followed by incubation with the medium at 37 ° C for 48 hours. The survival rate of the cells was examined by an MTT (Invitrogen) test. The cells were cultured in a medium containing 0.1 mg/ml of MTT, and cultured at 37 ° C for 2.5 hours. The formazan crystals were then dissolved in DMSO (Sigma-Aldrich), and the absorbance was measured at 540 nm using a microplate analyzer (Model 680, BioRad). Each analysis needs to be repeated three times. The data presented is the percentage of viable cells compared to cells in the untreated control group.

In vitro apoptosis study

H1993 cells were seeded in 24-well plates, cultured overnight to attach them, and then cultured in 2.5 μg/ml Ms20-LD or LD in RPMI 1640 containing 10% FBS, respectively. After 24, 48 and 72 hours of incubation with the drug, the cells were harvested in lysis buffer (Cell Signaling) and subjected to Western blot analysis. The antibodies used were as follows: rabbit anti-cleaving caspase 9 (Asp 315), rabbit anti-cleaving caspase 3 (Asp 175), rabbit anti-PARP (both purchased from Cell Signaling), and murine anti-α-tubulin (Sigma) -Aldrich). Chemical fluorescence signals were detected using the BioSpectrum 600 Imaging System (UVP) and quantified in Vision WorksLS software (UVP).

Synthesis of scFv-bound quantum dots (QD)

In vitro cell binding assays and in vivo imaging assays were performed with Qdot 705 and Qdot 800 ITK amine PEG quantum dots (Invitrogen), respectively. The procedure for synthesizing the ligand-binding QD was modified with reference to previous studies (Cai and Chen (2008) Nat Protoc 3:89-96). Briefly, QD is combined with a sulfonic acid-SMCC (sulfo-succinimide-4-(N-methyleneimineiminemethyl)cyclopentane-1-carboxylate; Pierce) to The maleic acid-activated surface was generated on the QD, followed by removal of the unbound sulfonic acid-SMCC by a NAP-10 desalination column (GE healthcare). Ms20 was reduced by TCEP to form an activated thiol group at the carboxy terminus, which was then incubated with the maleimine-functionalized QD overnight at 4 °C. The Ms20-bound QD was purified by gel filtration (sepharose) 4B gel filtration chromatography in HEPES buffer. The concentration of the QD was analyzed by a spectrofluorometer and was calculated by interpolation using a standard curve.

In vivo fluorescent images of human tumor xenografts

Six 12-week old SCID male mice were subcutaneously transplanted with 2 x 10 ⁶ H1993 cells. When the size of the tumor reached approximately 300 mm ³ , the mice were randomized into two groups (3 mice per group) and injected intravenously with 400 pmole Ms20-QD or QD. When the mice were anesthetized with isofluoran, the Xenogen IVIS 200 imaging system (excitation: 525/50 nm; emission: 832/65 nm) was used to perform fluorescence imaging at the indicated time. After the filming, the rats were sacrificed by cervical dislocation. Organs and tumors were removed from the mice and analyzed by fluorescence imaging. In order to quantitatively compare the accumulation of Ms20-QD and QD in the tumor, the fluorescence intensity was calculated by removing the background value by Xenogen.

Animal model of phage target test in vivo

The six SCID mice (six weeks of age) at 5 × 10 ⁶ cells were injected subcutaneously of lung cancer. When the tumor reached a size of about 300 mm ³ , the mice were randomly divided into two groups (3 mice per group) and 2 × 10 ⁹ colony forming units (cfu) of anti-c-Met scFv phage selection strain 20 or control group. The phage is administered intravenously. After perfusion in 50 ml PBS, the organs and tumor tissues were removed, washed with iced PBS, and weighed. The phage bound to the tumor tissue was recovered as TG1 in growth, and the eluted phage valence was measured.

The tissue distribution of the target phage in mice containing human tumors was evaluated by immunohistochemistry using a highly sensitive polymer-HRP IHC detection system (BioGenex). Paraffin-embedded tissue samples were stained with mouse anti-M13 antibody followed by Super Enhancer reagent and polymer horseradish peroxidase-labeled anti-mouse IgG ) to work. After washing with PBST (0.1% Tween 20 in PBS), the sections were immersed in a solution of 3,3'-diamino-benzidine (DAB) supplemented with 0.01% H ₂ O ₂ and rinsed with PBST. The tissue sections were comparatively stained with hematoxylin and fixed with 50% glycerol in PBS, followed by analysis with an erect microscope (Axioplan 2 Imaging MOT, Zeiss). Animal care is carried out in accordance with the norms of the Taipei Institute of Chinese Studies.

Animal model for measuring the anti-tumor effect of scFv target therapy

SCID mice containing H460-derived lung cancer xenografts (~75 mm ³ ) were given a total doxorubicin dose of 4 mg/kg (1 mg/kg once a week), and Ms20-LD was injected intravenously into the tail. LD or equivalent PBS,. Tumors were measured with calipers twice a week, and the body weight loss of the rats was regularly observed as a symptom of drug toxicity. Tumor volume is calculated according to the formula: length x (width) ² x 0.52.

In situ terminal deoxynucleotidyl transferase dUTP labeling (TUNEL) assay

Tumors obtained from mice in the treatment group were embedded in liquid nitrogen with O.C.T compound (Tissue-Tek). Frozen tumor tissue sections were prepared and treated with TUNEL reagent according to the manufacturer's instructions (Roche Diagnostics) and contrast stained with DAPI. A full-slice scan was performed with a TissueFAXS system microscope (TissueGnostics). Cell viability identified all cells by setting DAPI as the primary microscopic channel and quantified by MetaMorph software (Molecular Devices).

Tumor vascular staining

After sections were prepared from frozen tumor tissues, the sections were fixed with 1% paraformaldehyde, rinsed with PBS, and blocked with normal horse serum (Vector Laboratories), followed by rat anti-rat Mouse CD31 (BD) effect. After washing with PBS containing 0.1% Tween 20, the sections were soaked with Alexa Fluor 594-conjugated anti-rat IgG (Invitrogen). The sections were then scanned with a TissueFAXS system microscope and the fluorescent images were analyzed by MetaMorph.

Example 1

Identifying phage that bind to c-Met and express scFv

In order to screen out scFvs that bind to c-Met, it is necessary to construct a human native scFv library containing 2 x ¹⁰⁹ types of phage expression. The phage that binds to Dynabeads is first removed first, and the phage that binds to c-Met is screened by c-Met-conjugated Dynabeads. After 5 rounds of affinity screening, the phage recovered in the fourth round had an affinity of about 1000 times higher than that recovered in the first round (Fig. 1A). The 5th phage selection strain was randomly selected and tested for binding ability to c-Met by ELISA. The phage colony of No. 14 was found to have excellent binding activity to the c-Met-Fc protein ( A ₄₉₀ >0.2). The control group phage (Con-P) was used as a negative control group. After identification, 16 strains were specifically bound to c-Met, but not to VEGFR2 and BSA-controlled histones (data not shown). All 16 colonies were sequenced and 3 unique anti-c-Met phage selectants (PC1, PC20, and PC21) were found. Please refer to Table 1 below.

Complementarity determining regions V _H and V _L regions of 1-3 (CDR1-3), and framework regions 1-4 (FW1-4) are presented in the table. The family of the V-zone is aligned by the VBASE2 database ( www.vbase2.org ).

To analyze the specificity and binding affinity of the three phage selection strains, competitive ELISA analysis was performed at the same phage cost. This PC1 has a stronger c-Met binding affinity compared to PC20 or PC21 (Fig. 1B). The 293T cells expressing human c-Met were ectopically analyzed by flow cytometry and immunofluorescence assays to analyze the binding of the three selected strains to c-Met on the cell surface. Phage particles were cultured with 293T cells which overexpress c-Met, followed by anti-M13 and anti-myc antibodies, respectively, to detect phage particles and exogenous c-Met. In the representative representation of Figure 1, Figure 1D shows that anti-c-Met phage can be colocalized with c-Met-myc, meaning that they can all specifically bind to cells that will express c-Met. The three selected strains only bound to 293T cells that would overexpress c-Met without binding to 293T cells (Figures 1C and D). However, similarly, PC1 has a higher binding ability to c-Met of cells than PC20 and PC21. Next, the kinetic constants of anti-c-Met scFvs were investigated to produce c-Met _932- Fc recombinant protein and soluble anti-c-Met scFvs, referred to as S1, S20 and S21, corresponding to PC1, PC20 and PC21. The soluble c-Met _932- Fc protein was purified from the culture medium of 293T cells expressing ectopic c-Met _932- Fc by a protein G gel tube (Fig. 8). The soluble anti-c-Met scFvs were obtained from the protoplast-like gap extract of Escherichia coli HB2151 infected with phage, purified by protein L agaric chromatography, and then analyzed by protein staining (coomassie blue staining). To obtain a purified anti-c-Met scFv protein (S1, S20 and S21) near the 30 kDa position (corresponding to the protein weight index) (Fig. 8B upper panel).

The binding kinetic constants ( K _on and K _off ) and the affinity of each of the soluble scFvs having affinity for the c-Met _932- Fc protein were measured by surface plasma resonance (SPR). It may bind to c-Met soluble scFv ₉₃₂ -Fc each of K _d values of lines between 6.82 to 14.9 nM. Please refer to Table 2 below.

K _on and K _off were measured by SRP at BIAcore using purified scFvs, and the K _d was calculated by BIAevaluation software.

Example 2

Anti-c-Met scFvs bind to human cancer cells and VEGF-stimulated endothelial cells

The binding ability of the anti-c-Met scFvs to endogenous c-Met was linked to cancer cells and analyzed by ELISA (Fig. 9A). Compared with the control group antibody, we found that anti-c-Met S1 and S20 can bind to a variety of human cancer cell lines, including SKOV3, Mahlavu, SAS, PC3, MDA-MB-231, HCT116, Paca-2, NPC-TW04, H1993 cells. Normal mouse IgG was used as a control group antibody. FACS analysis was performed to assess whether the two anti-c-Met scFvs could also bind to VEGF-stimulated HUVECs. The anti-CD31 antibody and the control group scFv were used as a positive control group and a negative control group, respectively (Fig. 9B).

In order to further confirm that the anti-c-Met scFv can specifically bind to the endogenous c-Met on human cancer cells, H460 cells and c-Met knockout H460 cells were stained with anti-c-Met scFv S1 or S20, FACS analysis was then performed (Fig. 10B). Both scFvs were able to bind to H460 cells, but their binding capacity on c-Met knockout H460 cells was significantly reduced. B-tubulin was used as a negative control, and *bkg on Figure 10A was the background signal.

Example 3

Competing with HGF for binding to cancer cells by anti-c-Met scFv

To test whether the three anti-c-Met scFvs could inhibit HGF binding to c-Met, human lung cancer cell line H1993 was selected for competitive ELISA (Lutterbach B et al. (2007) Cancer Res 67:2081-2088). Under the same conditions, the control group phage (Con-P) did not affect the binding ability. HGF binds to H1993 cells and is considered 100% if there is no competitor. H1993 cells are known to exhibit high amounts of c-Met. When H1993 cells were cultured with HGF and phage-selected strains, the binding ability of HGF to H1993 cells was reduced by more than 90% by anti-c-Met scFv PC1. PC20 inhibited the binding capacity of more than 50% of HGF binding to c-Met (Fig. 2A). Competition inhibition of different concentrations of soluble scFvs for HGF was also tested. Normal mouse IgG (NM IgG) was used as a negative control group. HGF binds to the cured c-Met protein, and is considered 100% when there is no competitor. As shown in Figure 2B, the binding activity of HGF to c-Met was a dose-dependent inhibitory effect for S1 and S20; however, it had only a slight inhibitory effect on S21. And S1 to S20 for the binding of HGF to c-Met IC ₅₀ were 27.4 nM and 249.5 nM (FIG. 2B).

Therefore, 遂 explores the binding epitope of anti-c-Met scFvs in the c-Met HGF binding region. The binding ability of the anti-c-Met scFvs was analyzed by c-Met _932- Fc protein, including the whole extracellular region of c-Met, and the c-Met _567- Fc protein containing Sema and PSI regions, which have been Defined as the HGF binding region (Figure 2C). The c-Met recombinant protein was immobilized on a microplate and allowed to act together with anti-c-Met scFvs. Anti-c-Met polyclonal antibodies and anti-E-labeled antibodies were used as positive and negative controls, respectively. As shown in Figure 2D, S21 significantly reduced its binding activity to c-Met _567- Fc compared to c-Met _932- Fc. The binding strength of S1 to c-Met _932- Fc is similar to that of c-Met _567- Fc. The efficiency of S20 binding to c-Met _567- Fc was lower than that of binding to c-Met _932- Fc. The results show that the binding epitopes of S1 and S20 are located in the HGF binding region of c-Met, wherein S21 can pass through the Ig-like region to recognize c-Met (Fig. 2D).

Since the anti-c-Met scFv S1 exhibited better competitiveness, it was analyzed whether S1 can antagonize HGF to activate c-Met of cancer cells. A549 cells were treated with HGF and S1 for 15 minutes at 37 °C. The total cell lysate was subjected to the Western blot method using an anti-phosphotyrosine c-Met antibody and an antibody against c-Metβ-chain (c-Met). Phosphorylated c-Met is quantified based on luminescence intensity and normalized with total c-Met. HGF-induced c-Met phosphorylation was inhibited by S1, and 65.7% of c-Met phosphorylation was inhibited by S1 treatment compared to those without S1 (Fig. 2E).

Example 4

Analysis of internalization of anti-c-Met scFv using conjugated focal microscope

The internalization ability of an antibody is an important factor for the development of antibody-mediated liposome drug lines (Sapra and Allen (2002) Cancer Res 62: 7190-7194). Internalization studies of anti-c-Met scFv S1 and S20 were performed on H1993 cells at 37 °C. The scFvs are detected by an anti-E-labeled antibody, and then cultured with FITC-labeled secondary antibody after cell fixation and pore formation. The nucleus was stained with DAPI. Conjugate focal microscopy showed that the scFvs bind to the cell membrane at 4 °C (Fig. 3A, a and b), and the fluorescent signal emitted by the internalized scFv in the cytoplasm at 37 °C (Fig. 3A, c and d) . The amount of fluorescent signal of H1993 cells treated with S20 was higher than that of cells treated with S1, which means that the internalization ability of S20 is superior to S1. At low magnification, internalized S20 was observed in most cancer cells (Fig. 3A, e).

Furthermore, in order to verify whether the absorption of S20 is dependent on the c-Met expressed on the cell surface, an experiment of internalization by H460 cells and c-Met-removed H460 cells was performed. Confocal focal microscopy showed S20 fluorescence signals in the cytoplasm of H460 cells (Fig. 3B, a and b), but there was no fluorescent signal on c-Met knockout H460 cells (Fig. 3B, c). These results indicate that anti-c-Met scFv S20 exhibits a specific internalization effect on cancer cells that will exhibit c-Met. As such, it can be used in the development of intracellular drug delivery methods for antibody vectors.

Example 5

Ms20-bound nanoparticle enhances drug binding, intracellular delivery, and cytotoxicity

In order to investigate whether S20 can express c-Met cancer cells and promote liposome drug delivery, a bacterial expression vector encoding S20 gene and cysteine at the C-terminus is constructed, and then the S20-cysteamine is expressed. The acid fusion protein, referred to herein as Ms20 (Figures 11A and B). The site-directed conjugation of Ms20 is transmitted through the C-terminal cysteine to bind to the maleimide-modified PEG chain on the outer surface of the microlipid containing doxorubicin to form Ms20-binding. The liposomal doxorubicin (Ms20-LD) (Figures 11C and D).

In order to confirm whether Ms20-bound microlipids would increase drug delivery to cancer cells, many human lung cancer cell lines were treated with Ms20-LD and LD at 37 °C. After being washed with an acidic glycine buffer, it can remove the liposome drug bound to the surface, ie, the internalized doxorubicin can be quantified. After treatment with Ms20-LD, the cellular uptake of doxorubicin was increased in H1993, H441 and A549 cells, but not in H520 and CL1-5 cells (Fig. 4A). To further verify whether the absorption of Ms20-LD is dependent on the c-Met performance of the cells, the relative c-Met performance of each cell was performed on a flow cytometer using Ms20-labeled quantum dots (Ms20-QD) (Fig. 4B). Interestingly, H520 and CL1-5 were found to exhibit only a very small amount of c-Met (Fig. 4B), which corresponds to the poor absorption capacity of these cells for Ms20-LD (Fig. 4A). This means that the absorption of Ms20-LD by cancer cells depends on the degree of expression of c-Met on the cell surface.

To verify that Ms20 does have the ability to increase the binding efficiency of LD to cancer cells, H1993 cells were incubated with different concentrations of Ms20-LD and non-standard LD (LD) for 1 hour at 4 °C. After the cells are to be lysed, the binding activity of the liposome drug is quantified by fluorescence. The ability of Ms20-LD to bind to H1993 cells increased significantly by 13 to 26 fold compared to LD, depending on the concentration used (Fig. 4C). Similar results were observed in H460 cells under the same conditions. To confirm that Ms20 can enhance intracellular drug delivery capacity for delivery to cancer cells, H1993 cells were incubated with Ms20-LD and LD for a period of time. After removing the liposome drug whose surface was not internalized, the cockroach was measured for the doxorubicin absorbed by internalization. At each time point, Ms20 significantly enhanced the ability of the drug to be delivered to cancer cells compared to those not in the LD (Figure 4D).

To further assess whether Ms20 can enhance the cytotoxicity of LD, the cytotoxic effects of Ms20-LD were studied by MTT assay in human lung cancer cells (Fig. 5A). Cell viability is calculated as a percentage of viable cells. The red dotted line refers to an average 50% survival rate. Each point represents the average of four experiments. Compared to the LD, Ms20-LD significantly enhance the cytotoxicity of cancer cells, and in H1993 and H441 cells, reducing the half maximal inhibitory concentration (IC ₅₀₎ about 6-fold (Figure 5B). The expression of these apoptosis indicators, such as cleavage of PARP, cleavage of caspase 9 and cleavage of caspase 3, were all enhanced in H1993 cells treated with Ms20-LD (Fig. 5C). The α-tubulin is used as a loading control. The folds of the cut PARP were estimated by densitometry compared to cells in the untreated group and normalized with alpha-tubulin (bottom panel).

Example 6

In vivo tumor targeting In vivo Tumor homing) and images of anti-c-Met scFv

To investigate the tumor-directing ability of anti-c-Met scFv in vivo, mice containing H460-derived lung tumor xenografts were injected intravenously with anti-c-Met scFv PC20 or control group phage. The tumor-derived PC20 is more expensive than the organ-derived viscera. The test was performed twice and all gave the same results. After perfusion, the bound phage was recovered and the price of the force derived from the tumor portion and the strength of the normal organ were determined. The results showed that the ability of anti-c-Met scFv PC20 to direct to the tumor was higher than that directed to normal organs (Fig. 6A). The control group phage has no such guiding ability. In addition, the tissue distribution of anti-c-Met scFv PC20 was analyzed by liquid-free staining of tissue sections with anti-phage antibodies. PC20 phage were found to be selectively distributed in tumor tissues, rather than normal organ tissues, such as brain, lung, and heart. These organs were obtained from PC20-treated mice, and in tumors and normal organ tissues. No control group phage was detected (Fig. 6B).

To test whether anti-c-Met scFv S20 can be applied to tumor imaging analysis, Ms20-bound quantum dots (Ms20-QD) or unbound quantum dots (QD) were injected into mice containing H1993-derived lung tumor xenografts. . The intensity of the near-infrared light (NIR) fluorescence signal was observed 6 hours after the injection, and the signal intensity of the cancer tissue of the Ms20-QD-treated mouse was 5.2 times higher than that of the QD-treated mouse group (Fig. 6C). Twenty four hours after the injection, the mice were sacrificed and dissected to analyze the tissue distribution of Ms20-QD. This representative image is the result of three independent experiments. As shown in Figure 6D, Ms20-QD can be efficiently and selectively accumulated in tumors relative to normal organs. The ability of Ms20-QD to direct tumors was 5.4 times higher than QD. These results show that anti-c-Met scFv can be used for imaging of tumors.

Example 7

Therapeutic effect of Ms20-LD on human lung cancer xenografts

To explore whether Ms20 can improve the efficacy of anticancer drugs, a target drug delivery system was formulated by combining Ms20 with polyethylene glycolated microlipid doxorubicin (Ms20-LD). SCID mice containing H460 xenografts (~75 mm ³ ) were injected intravenously with a total doxorubicin dose of 4 mg/kg (once a week, 1 mg/kg). Mice containing H460-derived lung cancer were administered Ms20-LD, LD, and PBS. The tumor size of the mice in the LD group and the PBS control group were 1.9 and 4.4 times (n=8)**, respectively, compared with the Ms20-LD group, P <0.01. The points in the graph represent the mean tumor volume. We found that the tumor volume of mice administered Ms20-LD was less than that of LD alone ( P < 0.01) (Fig. 7A). On day 25, the tumor size of the LD group was gradually greater than that of the Ms20-LD group by 1.9 times. There was no significant change in body weight of the Ms20-LD and LD groups during treatment (Fig. 7B). At the end of treatment, the average tumor weight of the mice treated with Ms20-LD was 0.31 g, which was 0.73 g compared to the LD-treated group; 1.8 g was injected into the PBS buffer group (Fig. 7C and D). Therefore, the tumor weight of the Ms20-LD group was lower than that of the LD group (n=8)*, P < 0.05. In addition, tumor tissues of each group were analyzed with anti-CD31 antibody to detect tumor blood vessels, and TUNEL analysis was used to identify apoptotic cells. Sections were analyzed by automated cell acquisition (Tissue Gnostics) and CD31 positive areas, and TUNEL positive areas were quantified by MetaMorph software (Molecular Devices). As shown in Fig. 7E, the CD31-positive region in the Ms20-LD treatment group was drastically reduced compared to the LD-treated group. Therefore, the amount of CD31-positive endothelial cells in the Ms20-LD group was lower than that in the LD group. The number of apoptosis in the Ms20-LD treated group was twice as high as in the LD treated group (Fig. 7F).

<110> Academia Sinica

<120> Anti-C-MET antibody and method of use thereof

<130> ACA0061WO

<150> 61/402,788

<151> 2010-09-03

<160> 53

<170> PatentIn version 3.5

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Figure 1 Screening and identification of phage that can express scFv that bind to the c-Met protein. A. To use a phage library that can express human native scFv to screen for phage (biopanning) that binds to the c-Met-Fc protein. B. Phage selection strains that bind to the c-Met-Fc protein will be screened and compared at two different ELISA rates. C. To compare the c-Met binding affinities of different phage selectants to cells, 293T cells overexpressed in c-Met were evaluated by flow cytometry. D. Determine the binding specificity of phage selectants by immunofluorescence staining. Scale bar: 50 μm.

Figure 2 is a competition test for c-Met binding with anti-c-Met scFv and HGF. A. By ELISA analysis, anti-c-Met scFv PC1, PC20 and PC21 expressed in phage expression were used to inhibit HGF, making it unable to bind to H1993 cells which would express c-Met. B. Dose-dependent inhibition of the ability of anti-c-Met scFv S1, S20 and S21 to bind HGF to c-Met protein by competitive ELISA assay. C. is a schematic representation of the human c-Met protein region. The Fc region of human IgG1 is fused to the carboxy terminus of c-Met ₉₃₂ and c-Met ₅₆₇ . D. Identification of anti-c-MetscFvs epitopes by using ELISA. E is an antagonistic effect on anti-c-Met scFv and HGF in cancer cells.

Figure 3 Analysis of internalization of anti-c-Met scFv by conjugated focus microscopy. A. H1993 cells were cultured with anti-c-Met scFv S1 and S20 at 4 ° C ( a and b ) for 30 minutes or at 37 ° C ( c and d ) for 30 minutes. At low magnification, most cells were observed to internalize S20 (e). The arrow indicates the boF scFv in the cells. B. Cells The S20 internalization system is transfected by c-Met. Wild-type c-Met (a) and c-Met knockout H460 cells (MET-KD) (c) were separately incubated with S20 at 37 ° C for 30 minutes. A high magnification field of view shows that a large amount of S20 is internalized into the cell (b).

Figure 4 Ms20 enhances the binding and internalization of the liposomal doxorubicin to human lung cancer cell lines. A. Internalization studies of Ms20-LD and LD in lung cancer cell lines, and the two were cultured at 37 ° C for 4 hours. B. Analysis of c-Met performance on the surface of cancer cells by flow cytometry using Ms20-QD. C. H1993 binds to liposome drug analysis. D. Absorption kinetics of the liposome drug. E. After incubation at 37 ° C for the time indicated by the illustration, Ms20-LD and LD were taken up by H1993 cells as observed under a conjugate focal microscope. After incubation with Ms20-LD for 2 hours, doxorubicin was distributed to the cytoplasm and nucleoplasm. After 8 hours of incubation with Ms20-LD, doxorubicin was apparently mainly accumulated in the nucleus. Doxorubicin is difficult to detect in such cells treated with LD. The figure below shows an image of the combination of the doxorubicin signal ( red ) with the cell membrane ( green, pseudo-color ) and the nucleus ( blue ) staining. The ruler is 50 μm.

Figure 5 Ms20 regulates liposomes to enhance doxorubicin-induced cell toxicity. A. In vitro cytotoxicity assay of human lung cancer cell lines treated with different concentrations of Ms20-LD and LD. B. IC ₅₀ was calculated scale factor for explaining the Ms20-LD enhanced cytotoxic potential and higher than the LD. C. H1993 cells were treated with Ms20-LD and LD at 2.5 μg/ml for 0, 24, 48 and 72 hours, respectively, and analyzed by Western blotting.

Figure 6 shows the ability of tumor targeting to anti-c-Met scFv in human lung cancer xenografts. A. SCID mice bearing human lung cancer H460 xenografts were injected intravenously with PC20 and control phage (Con-P). B. In the orientation test, the location of PC20 was analyzed by immunohistochemical staining. C. SCID mice bearing H1993 human lung tumors, in vivo ( in vivo ) images after intravenous injection of 400 pmole of Ms20-QD (quantum dot) (right) or QD (left). The NIR fluorescence image was taken 6 hours after the injection (top panel). The red circle indicates the location of the tumor. The signal intensity of the tumor area is quantified by IVIS software (below). D. The tissue distribution of Ms20-QD and QD was determined 24 hours after injection. The mouse was sacrificed and the organ was taken for NIR imaging (above). The signal intensity of tumors and organs is measured by IVIS software (below).

Figure 7 Effect of Ms20-LD on human lung cancer xenografts. A. L460-derived lung cancer mice were administered Ms20-LD, LD, or PBS, and their tumor volumes were analyzed. B. Body weight of each group. C. The weight of the tumor at the end of treatment. D. A representative map of the analysis described in Figure C. E. Explore the tumor blood vessels of tumor tissue. F. Analysis of apoptotic cells in the tumor area by the TUNEL assay. Error bar, SE.*, P <0.05.

Figure 8. Purification of soluble c-Met _932- Fc protein and anti-c-Met scFv. A. Comassie blue staining (left panel), and Western blot analysis using the anti-c-Met polyclonal antibody (right panel). The first lane is the total cell culture medium, the second lane is the passage through the protein G column column, and the third lane is the purified soluble c-Met _932- Fc protein. B. Soluble anti-c-Met scFvs were purified from the protoplast surrounding extract of phage infected E-coli HB2151. Western blot analysis showed that the soluble scFvs were recognized by anti-E-labeled antibodies (bottom panel).

Figure 9. Discussion on the binding ability of anti-c-Met scFvs to different human cancer cell lines and vascular endothelial cells (HUVECs). A. Anti-c-Met scFv S1 or S2 against ELISA results for different human cancer cell lines. B. The ability of anti-c-Met scFvs to bind to HUVECs was analyzed by flow cytometry.

Figure 10 Anti-c-Met scFvs specifically bind to endogenous c-Met in human lung cancer cells. A. Western blot analysis showed that c-Met in H460 cells (MET-KD H460 cells) was down-regulated after Lentivirus infection with c-Met shRNA. B. FACS analysis of the case where anti-c-Met scFvs bind to c-Met wild type and c-Met knocked out H460 cells.

Figure 11 Synthesis of Ms20-bound microlipid doxorubicin (Ms20-LD). A. The prokaryotic vector pFHC-S20 was constructed to express a schematic diagram of the scFv protein (Ms20) containing a Flag tag, six histidine acids (6xHis), and a cysteine residue at the carboxy terminus. B. Ms20 purified by Ni ⁺ NTA gel (sepharose) and protein A agaric chromatography was subjected to SDS-PAGE analysis and protein staining. PPE refers to the protoplast surrounding extract; FL refers to the passage of the flow. C. is a schematic pattern showing the binding process of reduced Ms20 to LD bound to maleimine-PEG-DSPE. D. Ms20-bound LD purified by gel filtration with gel 4B by SDS-PAG analysis and silver nitrate staining. Lanes 3-8 are Ms20 (upper band) after binding to maleimine-PEG-DSPE.

Figure 12 shows the expression of c-Met in human lung cancer cell lines by FACS analysis with MS20-QD. A. Human lung cancer cell lines were incubated with 10 μM Ms20-QD and QD for 1 hour at 4 °C. FACS analysis is used to assess binding capacity. B. H1993 cells were incubated with 50 nM Ms20-QD for 30 minutes at 37 °C.遂 The conjugated focal microscope was used to analyze the binding and absorption of Ms20-QD and H1993 cells. Ruler 50 μm.

Claims (11)

An isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable region ( _VH ) comprising a complementarity determining region (CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOS: 14 , 16 and 25 of the amino acid sequence, and a light chain variable region (V _L), which is complementary to the light chain variable region comprising CDR1, CDR2, and CDR3 sequences, respectively, comprising SEQ ID NOS: 35,37 and 46 of Amino acid sequence. An isolated antibody or antigen-binding fragment thereof according to claim 1 is a single-chain Fv (scFv), IgG, Fab, (Fab') ₂ , or (scFv') ₂ . An isolated antibody or antigen-binding fragment thereof, as claimed in claim 1 or 2, binds to an anti-cancer agent or a tag. A lipid nanoparticle comprising a surface and an internal space, the internal space comprising an anticancer agent, wherein the isolated antibody or antigen-binding fragment thereof according to claim 1 or 2 is attached thereto The surface of the lipid nanoparticle. The lipid nanoparticle according to claim 4, wherein when the lipid nanoparticle is contacted with a cell which exhibits cell surface c-Met, the antibody binds to the cell surface c-Met, and the lipid nanoparticle Being sucked. A composition comprising: a pharmaceutically acceptable carrier, and an isolated antibody as described in claim 1 or 2, or a lipid nanoparticle as in claim 4 or 5. The composition of claim 6, wherein the composition is formulated to be administered parenterally. The composition of claim 6 or 7 can be used for cancer treatment, diagnosis, drug delivery, and visualization. A pharmaceutical composition for treating a patient having cancer, comprising: the antibody according to claim 1 or 2, or the lipid nanoparticle according to claim 4 or 5, wherein the amount Is enough to slow down cancer Growing. The pharmaceutical composition of claim 9, wherein the cancer is lung cancer. An isolated nucleic acid comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of: (i) a heavy chain variable region ( _VH ), the heavy chain variable region comprises CDR1, CDR2, and CDR3 sequences, such sequences comprising SEQ ID NOS: 14, 16 and 25 of the amino acid sequence; and (ii) a light chain variable region (V _L), the light chain variable The region comprises complementary CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOS: 35, 37 and 46, respectively.